Methods for identifying therapeutic targets and treating and monitoring cancers

ABSTRACT

The present invention provides methods for treating cancers having a mutation in one or more tumor suppressor genes, comprising providing to a subject in need thereof an inhibitor of a kinase, as well as related methods and compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 16/859,663, filed on Apr. 27, 2020, which is a divisional ofU.S. Non-Provisional application Ser. No. 15/038,342, filed on May 20,2016, now granted U.S. Pat. No. 10,676,746, issued on Jun. 9, 2020,which is a U.S. National Phase Application of International PatentApplication No. PCT/US2014/066884, filed Nov. 21, 2014, now expired;which claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/907,943, filed on Nov. 22, 2013, now expired, each ofwhich is incorporated by reference herein in its entirety.

STATEMENT REGARDING GOVERNMENT INTEREST

This invention was made with government support under CA176303,CA217883, and CA214428 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is FHCR_028_03US_ST25.txt. The text file is 21 KB,was created on Jan. 25, 2021, and is being submitted electronically viaEFS-Web.

FIELD OF THE INVENTION

This invention relates to methods of treating cancers associated withmutation of the p53 gene, including but not limited to head and necksquamous cell carcinomas (HNSCC), as well as methods of monitoring theprogression of cancers and the response of cancers to treatment. Inaddition, the invention related to methods of identifying therapeutictargets and therapeutic agents for the treatment of cancers and otherdiseases.

BACKGROUND OF THE INVENTION

Patients with head and neck squamous cell carcinoma (HNSCC) are treatedaggressively with surgery followed by radiation, often together withcisplatin (1). Although these treatments increase loco-regional control,they are frequently disfiguring and induce high-grade toxicitieslimiting their effectiveness (2). Furthermore, resistance to cisplatinand radiation contributes to tumor recurrence, and options for those whodo not respond are limited to palliative care. Targeted therapies forHNSCC are scarce, limited to experimental agents targeting the epidermalgrowth factor receptor (3).

Mutations in the tumor suppressor gene p53 are very common in HNSCC,with an estimated frequency of >50% (4, 5). Disruptive p53 mutationshave been associated with metastasis, resistance to radiation, and poorpatient survival (6-8). Despite the strong implication of p53 in thebiology and clinical outcome of HNSCC, there are no available therapiesthat specifically target p53 mutant cancer cells.

The present invention addresses this shortcoming by providing newtherapies for the treatment of p53 mutant cancer cells, including HNSCC,as well as new methods for identifying therapeutic targets in cancersand other diseases.

BRIEF SUMMARY OF THE INVENTION

The present invention provides, inter alia, methods of treating andpreventing diseases, including cancers, as well as related methods ofmonitoring the progression of a disease, and methods of identifyingtherapeutic targets and therapeutic agents for use in treating orpreventing disease.

In one embodiment, the present invention includes a method for treatingor preventing a cancer in a subject in need thereof, wherein the cancercomprises a mutation in a tumor suppressor gene, the method comprisingproviding to the subject an inhibitor of a G2/M, SFK, PI3K or FAKpathway. In particular embodiments, the tumor suppressor gene is aDNA-PKcs, Atm, p19^(Arf), p53, Hras, Kras or Prkdc gene, or wherein thecancer comprises a mutation in both a p53 tumor suppressor gene and oneor more of a Hras or Kras gene. In one embodiment, the tumor suppressorgene is a p53 gene. In various embodiments, the inhibitor inhibits theexpression of a gene in the G2/M, SFK, PI3K or FAK pathway. In certainembodiments, the gene encodes a kinase. In certain embodiments, thekinase is listed in FIG. 1C, FIG. 7, or FIG. 8.

In particular embodiments, the inhibitor inhibits an activity of aprotein in the G2/M, SFK, PI3K or FAK pathway. In certain embodiments,the protein is a kinase. In particular embodiments, the kinase functionsin the G2/M transition. In certain embodiments, the kinase is listed inFIG. 1C, FIG. 7, or FIG. 8. In certain embodiments, the inhibitor is aG2/M checkpoint inhibitor. In one embodiment, the G2/M checkpointinhibitor is MK-1775. In certain embodiments, the kinase inhibitor isMK-1775, TAE684, PI828, PIK93, PP2, PF-562271, or AZD7762. In particularembodiments, any of these methods further comprise providing to thesubject cisplatin. In certain embodiments, the kinase is NEK4, WEE1,ILK, CAM2 KB, FGFR3, FYN, PI4 KB, TRIB2, TTK or TXK. In certainembodiments, the kinase is WEE1, ALK, PI3K, PIK4CB, FYN, or FAK. Incertain embodiments, the kinase is WEE1, NEK4 or AURKA. In certainembodiments, the kinase is WEE1, CHEK1, GK2, PIP5K1B, EPHA2, RPS6KL1,MPP3, EPHA3, or AURKA. In one embodiment, the kinase is WEE1 and theinhibitor is MK-1775 or 681640. In one embodiment, the kinase is ALK andthe inhibitor is TAE684. In one embodiment, the kinase is PI3K and theinhibitor is PI828. In one embodiment, the kinase is PIK4CB and theinhibitor is PIK93. In one embodiment, the kinase is FYN and theinhibitor is PP2. In one embodiment, the kinase is FAK and the kinaseinhibitor is PF-562271. In particular embodiments, the inhibitorcomprises a small molecule, a polynucleotide, or a polypeptide. Incertain embodiments, the polynucleotide is an antisense RNA, an siRNA,or an miRNA. In certain embodiments, the polypeptide is an antibody orfunctional fragment thereof.

In a related embodiment, the present invention includes a method fortreating or preventing a cancer in a subject in need thereof, whereinthe cancer comprises a mutation in a tumor suppressor gene, the methodcomprising providing to the subject an inhibitor of a kinase selectedfrom NEK4, TRIB2, TXK, CAMK2B, NUAK2, EPHA3, ALK, PIP5K1B, RPS6KL1,FGFR3, WEE1, and PRKCE.

In particular embodiments, the methods further comprise determining alevel of expression or activity of a kinase gene or its encoded geneproduct in cancer cells obtained from the subject. In some embodiments,determining the level of expression comprises determining an amount ofmRNA expressed by the kinase gene. In other embodiments, determining thelevel of expression comprises determining an amount of polypeptideencoded by the kinase gene. In particular embodiments, the kinase geneis listed in FIG. 1C, FIG. 7 or FIG. 8. In particular embodiments, thelevel of expression or activity of the kinase gene or its encoded geneproduct is determined prior to providing the subject with the inhibitor.In other embodiments, the level of expression or activity of the kinasegene or its encoded gene product is determined at about the same time asproviding the subject with the inhibitor. In other embodiments, thelevel of expression or activity of the kinase gene or its encoded geneproduct is determined after providing the subject with the inhibitor.

In another embodiment, the present invention includes a method ofmonitoring the progression or regression of a tumor in response to atreatment, comprising: (a) determining a level of expression or activityof a kinase gene or its encoded gene product in cancer cells obtainedfrom the subject prior to or at about the same time as providing thesubject with the inhibitor; and (b) determining a level of expression oractivity of a kinase gene or its encoded gene product in cancer cellsobtained from the subject following a time period after providing thesubject with the inhibitor; wherein a reduced level of expression oractivity determined for (b) as compared to the level of expression ofactivity determined for (a) indicates that the treatment is effective.In particular embodiments, the kinase gene is listed in FIG. 1C, FIG. 7or FIG. 8.

In a further related embodiment, the present invention includes a methodof identifying a therapeutic drug target in a diseased cell, comprising:(a) screening a plurality of siRNAs for their ability to inhibit growthor reduce viability of a human diseased cell or a human cell having adefined mutation; (b) identifying one or more genes targeted by one ormore siRNAs of (a) that inhibit growth or reduce viability of the humancell; (c) screening a plurality of siRNAs for their ability to inhibitgrowth or reduce viability of a non-human diseased cell or a non-humancell having a defined mutation; (d) identifying one or more genestargeted by one or more siRNAs of (c) that inhibit growth or reduceviability of the non-human cell; and (e) determining one or more genesidentified according to both (b) and (d), wherein the human diseasedcell and the non-human disease cell share the same disease or themutation in the human cell is in the same gene as the mutation in thenon-human cell, and wherein the one or more genes determined accordingto (e) or their encoded products are identified as therapeutic drugtargets. In certain embodiments, the pluralities of siRNAs targetkinases. In certain embodiments, the non-human cell is a mammalian cell.In particular embodiments, the diseased cell is a tumor cell, e.g., aprimary tumor cell or a metastic or relapsed tumor cell. In particularembodiments, the cell comprises a mutation in a tumor suppressor gene,e.g., a DNA-PKcs, Atm, p19^(Arf), p53, or Prkdc gene.

In a further embodiment, the present invention includes a method fortreating or preventing a cancer in a subject in need thereof,comprising: (a) screening a plurality of siRNAs for their ability toinhibit growth or reduce viability of a cancer cell obtained from thesubject; (b) identifying one or more genes targeted by one or moresiRNAs of (a) that inhibit growth or reduce viability of the cancercell; and (c) providing to the subject an inhibitor of one or more ofthe genes identified in (b). In certain embodiments, the method furthercomprises obtaining a biological sample comprising a cancer cell fromthe subject. the plurality of siRNAs target kinases.

In yet another related embodiment, the present invention includes amethod for treating or preventing a cancer in a subject in need thereof,comprising: (a) obtaining or requesting the results of a testcomprising: (i) screening a plurality of siRNAs for their ability toinhibit growth or reduce viability of a cancer cell obtained from thesubject; and (ii) identifying one or more genes targeted by one or moresiRNAs of (a) that inhibit growth or reduce viability of the cancercell; and (b) providing to the subject an inhibitor of one or more ofthe genes identified according to (a). In particular embodiments, theplurality of siRNAs target kinases.

In particular embodiments of any of the methods described herein, thecancer is a squamous cell carcinoma, e.g., a head and neck squamous cellcarcinoma. In particular embodiments, the cancer is a p53 mutantsquamous cell carcinoma. In certain embodiments, the tumor is a primarytumor, while in other embodiments, the tumor is a recurrent ormetastatic tumor. In particular embodiments, the cancer is a head orneck cancer, a breast cancer, a prostate cancer, a brain cancer, athyroid cancer, a lung cancer, an ovarian cancer, a stomach cancer, apancreatic cancer, a liver cancer, a skin cancer, a leukemia, alymphoma, a colon cancer, a cervical cancer, a uterine cancer, anesophageal cancer, or a bladder cancer.

In particular embodiments, of any of the methods described herein, theinhibitor inhibits one of the following kinases: NEK4, WEE1, ILK, CAM2KB, FGFR3, FYN, PI4 KB, TRIB2, TTK or TXK. In certain embodiments, thekinase is WEE1, ALK, PI3K, PIK4CB, FYN, or FAK. In certain embodiments,the kinase is WEE1, NEK4 or AURKA. In certain embodiments, the kinase isWEE1, CHEK1, GK2, PIP5K1B, EPHA2, RPS6KL1, MPP3, EPHA3, or AURKA.

In various embodiments of any of the methods of treatment describedherein, the inhibitor is provided to the subject in combination withanother therapeutic agent.

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C provide diagrams showing the comparative functional kinomicsapproach to identify survival kinases in squamous cell carcinoma. FIG. 1A. Flow chart schematic of functional kinomic approach for discovery ofkinase targets. RNAi screen hits from both murine (MSCC) and human(HNSCC) cells were prioritized by cross-species comparison. FIG. 1 B.Cartesian plots of Zscores from interspecies kinome (508 kinases) toidentify kinase targets which had the highest shared impact on cellviability. Human and mouse cell lines were sorted into each of threecomparisons: all cells (left), p53 mutant cells (middle), and metastaticcells (right). Candidate kinase targets with Zscores greater than onestandard deviation from the mean cell viability per comparison are shownin gray. FIG. 1C. Venn diagram of selection of 38 kinase targets frominterspecies comparison; inclusion in diagram represents kinases targetsthat met a certain threshold in each comparison.

FIGS. 2A-2K provide bar graphs showing the results of RNA interferencevalidation screens on kinase targets. FIGS. 2A-2D. RNAi primaryvalidation screen; left bar graphs show differential cell viability (day4.5−day 1.5) of RNAi-mediated knockdown of 28 kinase targets inautologous pairs of HNSCC cell lines derived from primary tumor andrecurrent/metastatic site (14A,14C,15A,15B); kinase target vs. UNI,P<0.05 (green); right bar graphs show caspase-dependent apoptosisintegrated over 4.5 day time-course of RNAi-mediated knockdown of 28kinase targets, kinase target vs. UNI, P<0.05 (blue); results ranked bydifferential viability for each kinase target per HNSCC cell line. Celllines assayed include UMSCC-14A (FIG. 2A); UMSCC-14C (FIG. 2B); PCI-15A(FIG. 2C); and PCI-15B (FIG. 2D). FIGS. 2E-2I. RNAi secondary validationassays in five additional HNSCC cell lines (22A, 22B, 38, 47, 019); leftbar graphs show differential cell viability (day 4.5−day 1.5) ofRNAi-mediated knockdown of 20 kinase targets in HNSCC cell lines, kinasetarget vs. SINC, P<0.05 (J); right bar graphs show caspase-dependentapoptosis integrated over 4.5 day time-course of RNAi-mediated knockdownof 20 kinase targets in HNSCC cell lines, kinase target vs. SINC, P<0.05(K), results ranked by differential viability for each kinase target perHNSCC cell line. Cell lines assayed include UMSCC-22A (FIG. 2E);UMSCC-22B (FIG. 2F); UMSCC-38 (FIG. 2G); UMSCC-47 (FIG. 2H); and JHU-019(FIG. 2I). FIG. 2J and FIG. 2K. Kinase target significance in HNSCC.RNAi-mediated knockdown of kinase targets ranked by percentage of HNSCCcell lines in which kinase target reached statistical significanceversus universal negative siRNA control. Differential viability (FIG.2J); caspase 3/7 dependent apoptosis (FIG. 2K).

FIGS. 3A-3D. Chemical inhibition of SRC family kinase (FYN),phosphatidylinositiol kinases (PIK3CB, PIK4CB), focal adhesion kinase(FAK), tyrosine kinase receptor (ALK), and G2/M mitotic kinase (WEE1)impairs viability of autologous pairs of p53 mutant HNSCC cells.Dose-response curves performed with six kinase inhibitors (MK-1775,TAE684, PF-562271, PI828, PIK93, PP2) against kinase targets (WEE1, ALK,PIK3CB, PIK4CB, FYN, FAK; 8-point, mean (N=3), range 100 μM-30 nM,R²>0.85 for all curves. Autologous HNSCC cell pairs (UMSCC-14A (FIG.3A), UMSCC-14C (FIG. 3B), PCI-15A (FIG. 3C), PCI-15B (FIG. 3D)) derivedfrom primary and recurrent/metastatic site from the same patient.

FIGS. 4A-4E. Drug sensitivity of MSCC and HNSCC cells to chemicalinhibition of G2/M mitotic kinases determined by loss-of-functionmutational status of p53. FIG. 4A Differential sensitivity of MSCCp53+/+ and p53−/− cells to WEE1 inhibitor MK-1775 (FIG. 4A) andCHK1/CHK2 inhibitor AZD7762 (FIG. 4B). 12-point dose response curves,mean+/−sem (N=3), range: 10004-0.3 nM, R2>0.95 for all curves. FIG. 4CDifferential sensitivity of HNSCC p53+/+ and p53 mutant cell lines toMK-1775.11-point dose response curves, mean+/−sem (N=3), range 100 mM-1nM, R2>0.86 for all curves. FIG. 4D Box plots of TP53 mutation statusvs. IC50 values following treatment with the dual WEE1/CHK1 inhibitor681640 based on data from the Genomics of Drug Sensitivity project. Leftpanel: comparison of TP53 wild type (n=8) and TP53 mutant (n=34) SCCcells. Right panel: comparison of TP53 wild type (n=177) and TP53 mutant(n=322) status across all cell lines excluding SCC lines, representing adiversity of tumor types. FIG. 4E Bar graph of MK-1775 IC₅₀ values foreach of the nine cell lines, where * indicates a statisticallysignificant difference in IC₅₀ values between p53 wild-type (WT) versuseither p53 wild-type, HPV(+) or p53-mutant HNSCC cell cells, ANOVA withthe Holm-Sidak posttest; *, P<0.05.

FIGS. 5A-5C. MK-1775 induces mitotic entry, polyploidy, and apoptosis inHNSCC p53 mutant cells. FIG. 5A Flow cytometric analysis of HNSCCp53+/+(UM-SCC17A) and p53 mutant (PCI-15B) cells showing % mitotic cellsat 8 hours post MK-1775 treatment (y-axis) vs. % cell death at 24 hours(x-axis) normalized to untreated cells. FIG. 5B Cell cycle profiles at24 hours post MK-1775 treatment demonstrates abrogation of G2 (4N) cellpopulation and generation of polyploidy in p53 mutant cells. FIG. 5CCaspase 3/7-dependent apoptosis in HNSCC p53+/+ and HNSCC p53 mutantcells over 48 hours at three different concentrations (30 nm, 100 nm, 1μM) of MK-1775 (x-axis); fold change AUC ratio=AUC [MK-1775]/AUC[Vehicle] (y-axis).

FIGS. 6A-6D. WEE1 inhibitor MK-1775 potentiates the efficacy ofcisplatin in established p53 mutant HNSCC xenografts. FIG. 6A Percentagegrowth of PCI-15B xenografts in mice treated with MK-1775 and/orcisplatin. Y-axis represents percentage change (mean+/−SEM) in tumorvolume over the course of a 4 week protocol for each group; n=7 pergroup; unpaired t-tests; control vs. MK1775, P=0.06; cisplatin vs.cisplatin plus MK1775, ** P=0.003. FIG. 6B Bioluminescence images of arepresentative tumor-bearing mouse from each group at start (Day 1) andend of protocol (Day 28). FIG. 6C Immunoblot of xenograft tumor lysatesfrom two vehicle and two MK-1775 treated mice with specific antibodiesto kinase substrates WEE1, pCdc2, Cdc2, p-Wee1, Wee1, and β-actinloading control. FIG. 6D Bar graph of relative protein levels ofimmunoblot analysis of WEE1, CDC2, p-CDC2, mean±SD; normalized valuesfor vehicle versus MK1775 were compared via the unpaired t test, P<0.05as significant; n=2; WEE1, ns; p=WEE1;*, P=0.02 (one tail); CDC2, ns;p-CDC2; *, P=0.04 (one tail).

FIG. 7. Primary validation siRNAs used for various target genes.

FIG. 8. Secondary validation siRNAs used for various target genes.

FIGS. 9A-9B. Plate layouts for RNA interference mediated knockdown in HTRNAI primary validation screen (FIG. 9A) and secondary RNAI validationassay (FIG. 9B). Drawings of 384-well format for primary validationscreen and 96-well format for secondary assay. Individual siRNAs andsiRNA pooled approach depicted along with replicate plates;CellTiter-Glo assay (Promega) and Apotox-Glo assay (Promega).

FIG. 10. Human foreskin fibroblast (HFF) kinase exclusion plot. The top38 kinase targets where evaluated to determine whether RNAi mediatedinhibition caused toxicity to normal cells utilizing kinome screeningdata on two HFFs primary cultures. The common lethal space is depictedin the left lower hand corner of the plot to indicate the kinases whoseinhibition caused >30% loss in cell viability in both HFFs screens.

FIGS. 11A-11C. Correlation plots of autologous HNSCC cell line pairsfrom primary tumor and recurrent/metastatic site from patients.Correlation plots were generated from differential viability profiles ofRNAi mediated knockdown of kinase targets. UM-SCC14A, UM-SCC14C[r=0.7354, P<0.0001] (FIG. 11A); UM-SCC15A, UM-SCC15B [r=0.6778,P<0.0001] (FIG. 11B); and UM-SCC22A, UM-SCC22B [r=0.6424, P<0.0013](FIG. 11C). Pearson correlation coefficients generated utilizing 30 datapoints on UMSCC14, UM-SCC15, and 22 data points on UM-SCC22.

FIG. 12. Body weight of MK-1775 treated mice. Bar graph of percentagechange (Mean+/−SEM) in body weight for all treatment groups; unpairedt-tests: control vs. MK-1775, non-significant (ns); cisplatin vs.cisplatin plus MK-1775, ns; control vs. cisplatin, ****P<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the development of a novelapproach to identify therapeutic target genes and biological pathwaysassociated with diseases, such as cancers, and the use of this approachto identify new therapeutic target genes and pathways in cancersassociated with p53 mutations, such as HNSCC.

As described in the accompanying Examples, a functional kinomicsapproach and human-murine interspecies comparison of high-throughputsiRNA viability screens were employed to identify conserved survivalpathways in squamous cell carcinoma, in order to address the unmet needto find novel therapies for p53 mutant head and neck squamous cell. Therationale for targeting kinases in cancer was significant, and as such,efforts were focused on the kinome to identify druggable and clinicallyrelevant survival kinases in HNSCC. These studies revealedvulnerabilities of p53 mutant HNSCC cells to inhibition of G2/M, SFK,PI3K and FAK pathways. Preclinical validation studies performed on thekinase target, WEE1, demonstrated proof of concept and mechanism.Preclinical data demonstrated the vulnerability of p53 mutant HNS CCcells to deregulation of G2/M transition, and support initiation ofclinical trials with MK-1775 or other G2/M checkpoint inhibitors for thetreatment of HNSCC, alone or in combination with cisplatin.

Definitions and Abbreviations

The words “a” and “an” denote one or more, unless specifically noted.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length. In any embodiment discussed in the context ofa numerical value used in conjunction with the term “about,” it isspecifically contemplated that the term about can be omitted.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

By “consisting essentially of” is meant including any elements listedafter the phrase, and limited to other elements that do not interferewith or contribute to the activity or action specified in the disclosurefor the listed elements. Thus, the phrase “consisting essentially of”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

An “increased” or “enhanced” amount is typically a “statisticallysignificant” amount, and may include an increase that is 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times)(including all integers and decimal points in between and above 1, e.g.,2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein.

A “decreased” or “reduced” or “lesser” amount is typically a“statistically significant” amount, and may include a decrease that isabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100,500, 1000 times) (including all integers and decimal points in betweenand above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or leveldescribed herein.

A “composition” can comprise an active agent, e.g., a kinase inhibitor,and a carrier, inert or active, e.g., a pharmaceutically acceptablecarrier, diluent or excipient. In particular embodiments, a compositionis sterile, substantially free of endotoxins or non-toxic to recipientsat the dosage or concentration employed.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

The terms “mammal” and “subject” includes human and non-human mammals,such as, e.g., a mouse, rat, rabbit, monkey, cow, hog, sheep, horse,dog, and cat.

“Pharmaceutically acceptable salts” include sulfate, citrate, acetate,oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, lsomcotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate,phenylacetate, trifluoroacetate, acrylate, chlorobenzoate,dimtrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate,o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate,alpha-hydroxybutyrate, butyne-1,4-dicarboxylate,hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate,heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate,mesylate, mcotinate, phthalate, teraphthalate, propiolate, propionate,phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate,chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate,methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts. Theterm “pharmaceutically acceptable salt” also refers to a salt of anantagonist of the present invention having an acidic functional group,such as a carboxylic acid functional group, and a base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium, hydroxides of alkaline earth metal suchas calcium and magnesium, hydroxides of other metals, such as aluminumand zinc, ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine,tributylamine, pyridine, N-methyl, N-ethylamine, diethylamine,triethylamine, mono-, bis-, or tris-(2-OH-lower alkylamines), such asmono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine, N-methyl-D-glucamine, and amino acids such asarginine, lysine, and the like. The term “pharmaceutically acceptablesalt” also includes a hydrate of a compound of the invention.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not.

“Pharmaceutical composition” refers to a composition or formulation ofan active agent, e.g., a kinase inhibitor, and a medium generallyaccepted in the art for the delivery of the biologically active agentsto mammals, e.g., humans. Such a medium may include any pharmaceuticallyacceptable carriers, diluents or excipients therefore.

“Small molecule” refers to natural or synthetic small organic orinorganic compounds having a molecular weight of more than 50 and lessthan about 2,500 Daltons.

“Therapeutically effective amount” refers to that amount of an activeagent, e.g., a kinase inhibitor, that, when administered to a mammal,preferably a human, is sufficient to effect treatment, as defined below,of a disease or condition in the mammal, preferably a human. The amountof an active agent that constitutes a “therapeutically effective amount”will vary depending on the compound, the condition and its severity, themanner of administration, and the age of the mammal to be treated, butcan be determined routinely by one of ordinary skill in the art havingregard to his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest, e.g., tissue injury, in a subject ormammal, e.g., a human, having the disease or condition of interest, andincludes: (i) preventing or inhibiting the disease or condition fromoccurring in the subject or mammal, in particular, when such subject ormammal is predisposed to the condition but has not yet been diagnosed ashaving it; (ii) inhibiting the disease or condition, i.e., arresting itsdevelopment; (iii) relieving the disease or condition, i.e., causingregression of the disease or condition; or (iv) relieving the symptomsresulting from the disease or condition. As used herein, the terms“disease,” “disorder,” and “condition” may be used interchangeably ormay be different in that the particular malady, injury or condition maynot have a known causative agent (so that etiology has not yet beenworked out), and it is, therefore, not yet recognized as an injury ordisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

Methods of Treating, Preventing and Monitoring Disease

As demonstrated in the accompanying Examples, certain methods of thepresent invention are used to identify therapeutic targets for adisease. Accordingly, the present invention also provides methods oftreating a disease based on the identification of a therapeutic target.In particular embodiments, the therapeutic target is overexpressed indisease cells. In certain embodiments, inhibition of the therapeutictarget results in reduced growth, proliferation or viability of thedisease cell. In particular embodiments, inhibition of the therapeutictarget results in death of the disease cell. In particular embodiments,inhibition of the therapeutic target induces apoptosis of the diseasecell.

In one aspect, the present invention provides methods for inhibiting thegrowth and/or proliferation of diseased cells, e.g., cancer cells,comprising contacting the cells with an inhibitor of a kinase or kinasepathway. In related embodiments, the present invention provides methodsfor reducing viability or inducing apoptosis of diseased cells, e.g.,cancer cells, comprising contacting the cells with an inhibitor of akinase or kinase pathway. In one embodiment, the diseased cell iscontacted in vitro. In another embodiment, the diseased cell iscontacted in vivo, e.g., in a mammalian subject. In particularembodiments, the kinase is any of those described herein, e.g., WEE1,ALK, PIK4CB, FAK, NEK4, AURKA, CHK1, or a SRC family member or relatedkinase, such as SFK, FYN, TXK or CAM2 KB. In particular embodiments, thekinase is NEK4, TRIB2, TXK, CAMK2B, NUAK2, EPHA3, ALK, PIP5K1B, RPS6KL1,FGFR3, WEE1 or PRKCE. In certain embodiments, the present inventionprovides methods for inducing apoptosis of cancer cells, comprisingcontacting the cells with an inhibitor of WEE1, NEK4 or AURKA. Inparticular embodiments, the disease cells are contacted with two or moreinhibitors of a kinase or kinase pathway, e.g., an inhibitor of WEE1 andan inhibitor of CHK1.

In another aspect, the invention provides a method of treating orpreventing a disease in a subject in need thereof, comprising providingto or administering to the subject an amount of a composition comprisingan effective amount of an inhibitor of a kinase or kinase pathway. Inparticular embodiments, the composition is a pharmaceutical compositionfurther comprising one or more pharmaceutically acceptable carriers,diluents or excipients. In particular embodiments, the subject has beendiagnosed with or identified as being at risk of having a cancer, e.g.,a squamous cell carcinoma. In particular embodiments, the subject hasbeen diagnosed with a tumor having a mutation in p53, or another gene inthe p53 pathway. In particular embodiments, the kinase is any of thosedescribed herein, e.g., WEE1, ALK, PIK4CB, FAK, NEK4, AURKA, CHK1, or aSRC family member or related kinase, such as SFK, FYN, TXK or CAM2 KB.In particular embodiments, the subject is provided with or administeredtwo or more inhibitors of a kinase or kinase pathway, e.g., an inhibitorof WEE1 and an inhibitor of CHK1. In particular embodiments of these andother methods of the present invention, the diseased cells are cancercells, or the subject is diagnosed with or considered at risk forcancer. In particular embodiments, the cancer cell is a primary tumor ora secondary or metastatic tumor. In certain instances, the tumor is asolid tumor; in other instances, the tumor is a liquid tumor. In certainembodiments, the cancer cells have a mutation in a tumor suppressorgene, e.g., a DNA-PKcs, Atm, p19^(Arf), p53, Hras, Kras, or Prkdc gene.In certain embodiments, a subject is considered at risk of cancer if thesubject has a mutation in a tumor suppressor gene, including any ofthose described herein, such as p53.

In certain embodiments, the cancer cells have a mutation in a p53 tumorsuppressor gene. In certain embodiments, the cancer cells have amutation in both a p53 tumor suppressor gene and one or more of a Hrasor Kras gene. p53 mutations have been associated with a number ofdifferent types of cancer, including but not limited to lung cancer,stomach cancer, breast cancer, colon cancer, liver cancer, prostatecancer, cervical cancer, uterine cancer, head and neck cancer,esophageal cancer, leukemia, lymphoma, ovarian cancer, and bladdercancer. Any of these and other types of cancer may be treated orprevented according to the methods described herein. In certainembodiments, the cancer is a head or neck cancer, a breast cancer, aprostate cancer, a brain cancer, a thyroid cancer, a lung cancer, anovarian cancer, a stomach cancer, a pancreatic cancer, a liver cancer, askin cancer, a leukemia, a lymphoma, a colon cancer, a cervical cancer,a uterine cancer, an esophageal cancer, or a bladder cancer. Inparticular embodiments, the cancer is a squamous cell carcinoma, e.g., ahead and neck squamous cell carcinoma. In certain embodiments, thecancer is a squamous cell carcinoma of the lung, cervix, skin orbladder.

In one embodiments, the present invention includes a method for treatingor preventing a cancer in a subject in need thereof, the methodcomprising providing to the subject an effective amount of a compositioncomprising an inhibitor of a G2/M, SFK, PI3K or FAK pathway. Inparticular embodiments, the cancer comprises a mutation in a tumorsuppressor gene, e.g., p53. In particular embodiments, the inhibitorinhibits expression of a gene in the G2/M, SFK, PI3K or FAK pathway. Incertain embodiments, the inhibitor inhibits an activity of a protein theG2/M, SFK, PI3K or FAK pathway.

In other embodiments of any of the methods described herein, the canceris human papilloma virus (HPV) positive. In certain embodiments, the HPVpositive cancer is p53 wild-type, while in certain embodiments, the HPVpositive cancer comprises a mutation in a tumor suppressor gene.

In particular embodiments, the disease cell, e.g., a tumor cell,comprises a mutation in p53, and the kinase is WEE1, CDKN1B, PRKCE, GK2,or FGFR3. Thus, in certain embodiments wherein the tumor cell comprisesa mutation in p53, the subject is treated using an inhibitor of WEE1,CDKN1B, PRKCE, GK2, or FGFR3. In particular embodiments, the tumor is asquamous cell carcinoma, e.g., a head and neck squamous cell carcinoma.In particular embodiments, the subject is treated with the inhibitor incombination with cisplatin.

In particular embodiments, the disease cell, e.g., a tumor cell,comprises a mutation in p53, and the kinase is WEE1, CHEK1, GK2,PIP5K1B, EPHA2, RPS6KL1, MPP3, EPHA3 or AURKA. Thus, in certainembodiments wherein the tumor cell comprises a mutation in p53, thesubject is treated using an inhibitor of WEE1, CHEK1, GK2, PIP5K1B,EPHA2, RPS6KL1, MPP3, EPHA3 or AURKA. In particular embodiments, thetumor is a squamous cell carcinoma, e.g., a head and neck squamous cellcarcinoma. In particular embodiments, the subject is treated with theinhibitor in combination with cisplatin.

In certain embodiments, the disease cell, e.g., a tumor cell, comprisesa mutation in p19^(Arf), and the kinase is GK2, MAP2K7RIPK1, PLK1, orPRKCE. Thus, in certain embodiments wherein the tumor cell comprises amutation in p19^(Arf), the subject is treated using an inhibitor of GK2,MAP2K7RIPK1, PLK1, or PRKCE. In particular embodiments, the tumor is asquamous cell carcinoma, e.g., a head and neck squamous cell carcinoma.In particular embodiments, the subject is treated with the inhibitor incombination with cisplatin.

In certain embodiments, the disease cell, e.g., a tumor cell, comprisesa mutation in Atm, and the kinase is AK1, STK22B, CLK1, PRKCE or GK2.Thus, in certain embodiments wherein the tumor cell comprises a mutationin Atm, the subject is treated with an inhibitor of AK1, STK22B, CLK1,PRKCE or GK2. In particular embodiments, the tumor is a squamous cellcarcinoma, e.g., a head and neck squamous cell carcinoma. In particularembodiments, the subject is treated with the inhibitor in combinationwith cisplatin.

In certain embodiments, the disease cell, e.g., a tumor cell, comprisesa mutation in both KRAS and p53, and the kinase is WEE1, CHEK1, CAMK2B,or GK2. Thus, in certain embodiments wherein the tumor cell comprises amutation in KRAS and/or p53, the subject is treated using an inhibitorof WEE1, CHEK1, CAMK2B, or GK2. In particular embodiments, the tumor isa squamous cell carcinoma, or a pancreatic cancer. In particularembodiments, the subject is treated with the inhibitor in combinationwith cisplatin.

In certain embodiments, the disease cell, e.g., a tumor cell, comprisesa mutation in KRAS, and the kinase is FGFR3, NUAK2, ABL1, NEK4, AURKA,WEE1 or CHEK1. Thus, in certain embodiments wherein the tumor cellcomprises a mutation in KRAS, the subject is treated using an inhibitorof FGFR3, NUAK2, ABL1, NEK4, AURKA, WEE1 or CHEK1. In particularembodiments, the tumor is a squamous cell carcinoma, or a pancreaticcancer. In particular embodiments, the subject is treated with theinhibitor in combination with cisplatin.

In certain embodiments of the methods described herein, the gene that isinhibited encodes a kinase, or the protein that is inhibited is akinase. In certain embodiments, the kinase is listed in FIG. 1, FIG. 2,FIG. 3, FIG. 7 or FIG. 8. In particular embodiments, the kinase isselected from serine/threonine kinase 32B (STK32B); aurora kinase A(AURKA); galactokinase 2 (GALK2); thymidine kinase 2 (TK2),mitochondrial NUAK family, SNF1-like kinase 2 (NUAK2);calcium/calmodulin dependent protein kinase II beta (CAMK2B); membraneprotein, palmitoylated 3 (MAGUK) (MPP3); serine/arginine rich proteinspecific kinase 3 (STK23); phosphatidylinositol-4-phosphate 5-kinase,type 1, beta (PIP5K1B); glycerol kinase 2 (GK2); ribosomal protein S6kinase-like 1 (RPS6KL1); phosphatidylinositol 4-kinase, catalytic, beta(PIK4CB); FYN oncogene related to SRC, FGR, YES (FYN); eukaryotictranslation-initiation factor 2 alpha kinase 3 (EIF2AK3); inhibitor ofkappa light polypeptide gene enhancer in B cells, kinase epsilon(IKBKE); Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog(FGR); mitogen-activated protein kinase kinase kinase 8 (MAP3K8);uridine-cytidine kinase 1-like 1 (UCKL1); tribbles homolog 2(drosophila) (TRIB2); eukaryotic translation-initiation factor 2 alphakinase 4 (EIF2AK4); TXK tyrosine kinase (TXK); EPH receptor 3 (EPHA3);anti-Mullerian hormone receptor, type II (AMHR2); Wee1 homolog (S.pombe) (WEE1); Chk1 checkpoint homolog (S. pombe) (CHK1);mitogen-activated protein kinase 13 (MAPK13); TTK protein kinase (TTK);fms-related tyrosine kinase 1 (FLT1); phosphoinositide-3-kinase,catalytic, beta polypeptide (PIK3CB); Integrin-linked kinase (ILK);anaplastic lymphoma receptor tyrosine kinase (ALK); NIMA (never inmitosis gene a)-related expressed kinase 4 (NEK4); WNK lysine deficientprotein kinase 4 (WNK4); c-abl oncogene 1, non-receptor tyrosine kinase(ABL1); v-erb-b2 erythroblastic leukemia viral oncogene homolog 3(avian) (ERBB3); mitogen-activated protein kinase kinase kinase 2(MAP3K2); fibroblast growth factor receptor 3 (FGFR3); or Protein kinaseC, Epsilon (PKCE). In certain embodiments, two or more of these genesare inhibited according to a method of the invention.

In particular embodiments, the kinase is WEE1, CDKN1B, PRKCE, GK2,FGFR3, GK2, MAP2K7RIPK1, PLK1, PRKCE, AK1, STK22B, CLK1, PRKCE or GK2.In particular embodiments, the kinase is NEK4, WEE1, ILK, CAM2 KB,FGFR3, FYN, PI4 KB, TRIB2, TTK or TXK. In certain embodiments, thekinase is WEE1, ALK, PI3K, PIK4CB, FYN, or FAK. In certain embodiments,the kinase is WEE1, NEK4 or AURKA. In certain embodiments, the kinase isWEE1.

In certain embodiments, the kinase functions in the G2/M transition,e.g., WEE1, NEK4, TTK, AURKA and CHK1. In particular embodiments, theinhibitor is a G2/M checkpoint inhibitor. One example of a G2/Mcheckpoint inhibitor is MK-1775. In other embodiments, the kinase isimplicated in focal adhesion and integrin signaling, e.g., CAMK2B, FYN,ILK, EPHA3, EIF2AK4, and TRIB2. In certain embodiments, the kinase isimplicated in phosphoinositide 3-kinase (PI3K) signaling, e.g., PIK4B,PIK3CB, PIP5K1B, TRIB2, FGFR3 and ALK. In certain embodiments, thekinase is implicated in SRC signaling, e.g., FYN, TXK and CAM2 KB.

In particular embodiments of methods of the present invention, theinhibitor is MK-1775, TAE684, PI828, PIK93, PP2, PF-562271, or AZD7762.In certain embodiments, the inhibitor is MK-1775. The method of claim22, wherein the kinase is WEE1 and the inhibitor is MK-1775 or 681640.

In certain embodiments, the kinase is ALK, and the inhibitor is TAE684.

In certain embodiments, the kinase is PI3K, and the inhibitor is PI828.

In certain embodiments, the kinase is PIK4CB, and the inhibitor isPIK93.

In certain embodiments, the kinase is FYN, and the inhibitor is PP2.

In certain embodiments, the kinase is FAK, and the kinase inhibitor isPF-562271.

In particular embodiments, the disease is a cancer comprising a p53mutation, or comprising both a p53 mutation and a mutation in one orboth of Hras or Kras, and the kinase is listed in FIG. 1, FIG. 2, FIG.3, FIG. 7 or FIG. 8. In particular embodiments, the kinase is NEK4,WEE1, ILK, CAM2 KB, FGFR3, FYN, PI4 KB, TRIB2, TTK or TXK. In certainembodiments, the kinase is WEE1, ALK, PI3K, PIK4CB, FYN, or FAK. Incertain embodiments, the kinase is WEE1, NEK4 or AURKA. In certainembodiments, the kinase is WEE1. In particular embodiments, the kinaseis FGFR3, NUAK2, ABL1, NEK4, AURKA, WEE1 or CHEK1. In certainembodiments, the kinase is WEE1, and the inhibitor is MK-1775 or 681640.In particular embodiments, the subject is treated with the inhibitor incombination with cisplatin.

In particular embodiments, the cancer is a head and neck cancer or asquamous cell carcinoma, e.g., a head and neck squamous cell carcinoma,and the kinase is listed in FIG. 1, FIG. 2, FIG. 3, FIG. 7 or FIG. 8. Inparticular embodiments, the kinase is NEK4, WEE1, ILK, CAM2 KB, FGFR3,FYN, PI4 KB, TRIB2, TTK or TXK. In certain embodiments, the kinase isWEE1, ALK, PI3K, PIK4CB, FYN, or FAK. In certain embodiments, the kinaseis WEE1, NEK4 or AURKA. In certain embodiments, the kinase is WEE1. Inparticular embodiments, the head and neck cancer comprises a p53mutation. In certain embodiments, the kinase is WEE1 and the inhibitoris MK-1775 or 681640.

In one embodiment, the method comprises treating or preventing a headand neck squamous cell carcinoma in a mammal, e.g., a human, in needthereof, comprising providing or administering to the mammal aneffective amount of a pharmaceutical composition comprising MK-1775. Incertain embodiments, the carcinoma comprises a p53 mutation. In certainembodiments, the mammal is also provided with an effective amount of apharmaceutical composition comprising paclitaxel.

In certain embodiments, methods of the present invention furthercomprise providing or administering to the subject another therapeuticagent, in addition to the inhibitor. The inhibitor and the othertherapeutic agent may be provided concurrently or one may be providedbefore or after the other. In particular embodiments wherein the diseaseis a cancer, the other therapeutic agent is, e.g., a chemotherapeuticagent or a genotoxic agent. In certain embodiments, the chemotherapeuticagent or genotoxic agent is cisplatin.

In certain embodiments of the method for treating or preventing acancer, e.g., a p53 mutant cancer, in a mammal, e.g., a human, in needthereof, the method comprises providing or administering to the mammalan effective amount of an inhibitor of WEE1 and an effective amount of agenotoxic agent, such as cisplatin. In particular embodiments, thecancer is a squamous cell carcinoma, e.g., an HNSCC or a pancreaticcancer. In one embodiment, the method comprises treating or preventing ahead and neck squamous cell carcinoma in a mammal, e.g., a human, inneed thereof, comprising providing or administering to the mammal aneffective amount of a pharmaceutical composition comprising MK-1775 andan effective amount of the genotoxic agent, e.g., cisplatin. In certainembodiments, the carcinoma comprises a p53 mutation. In a particularembodiment, the method comprises administered to a subject having ap53-mutant cancer, e.g., a squamous cell carcinoma, such as a HNSCC,cisplatin in combination with an inhibitor of WEE1, such as MK-1775. Inparticular embodiments, such methods further comprising performingsurgery and/or treating the subject with radiation therapy. Inparticular embodiments, the method further comprises determining thatthe subject has a mutation in a tumor suppressor gene.

In a related embodiment, the present invention includes a method ofreducing the toxicity of cisplatin, comprising administering to asubject in need thereof cisplatin in combination with an inhibitor of akinase as described herein, e.g., an inhibitor of WEE1, such as MK-1775.In particular embodiments, the cancer comprises a mutation in a tumorsuppressor gene, e.g., p53. In particular embodiments, the methodfurther comprises determining that the subject has a mutation in a tumorsuppressor gene.

In a related embodiment, the present invention includes a method ofincreasing the efficacy of cisplatin, comprising administering to asubject in need thereof cisplatin in combination with an inhibitor of akinase as described herein, e.g., an inhibitor of WEE1, such as MK-1775.In particular embodiments, the cancer comprises a mutation in a tumorsuppressor gene, e.g., p53. In particular embodiments, the methodfurther comprises determining that the subject has a mutation in a tumorsuppressor gene.

In certain embodiments of any of the methods described herein comprisingthe administration of cisplatin, the amount of cisplatin administered isless than the amount recommended for treatment using cisplatin withoutthe kinase inhibitor. In particular embodiments, the amount of cisplatinadministered in a single treatment or administration is less than 100mg/m², less than 50 mg/m², less than 20 mg/m², less than 10 mg/m², lessthan 5 mg/m2, less than 1 mg/m², less than 0.5 mg/m², less than 0.2mg/m2, less than 0.1 mg/m², or less than 0.05 mg/m², or in any rangethat may be derived between these values. In certain embodiments of anyof the methods described herein comprising the administration ofcisplatin, the amount of cisplatin administered is a normal amountrecommended for treatment using cisplatin without the kinase inhibitor,while in other embodiments, it is a reduced amount, including any ofthose amounts described above.

In certain embodiments of any of the methods described herein, thesubject may also be treated via surgery and/or radiation therapy.

In some embodiments, the methods further comprise the step ofdetermining whether the tumor in the subject comprises a mutation in atumor suppressor gene, e.g., a p53 mutation, before providing to thesubject an inhibitor. The step of determining whether the tumor in thesubject comprises a mutation in a tumor suppressor gene, e.g., a p53gene, may be carried out by accessing a database, requesting the resultsof appropriate diagnostic or genetic testing, or by assaying cellsobtaining from the subject (such as a biopsy sample from said subject)for the presence or absence of a p53 mutation, using standard methodsknown in the art and as further described herein. A variety of specificp53 mutations associated with cancers are known in the art and may bedetected using routine methods. For example, a subject's p53 gene may besequenced, or probes specific for known mutations may be used toselectively amplify sequences comprising the mutations. In particularembodiments, a subject is treated with the inhibitor if the subject'stumor has the mutation in the tumor suppressor gene, and in otherembodiments, the subject is not treated with the inhibitor if thesubject does not have the mutation in the tumor suppressor gene, e.g.,p53. In certain embodiments of any of the methods herein that comprisedetermining whether a subject has a mutation in a tumor suppressor gene,such as p53, the subject is administered the inhibitor if the subjecthas said mutation but not if the subject does not have said mutation.Accordingly, in certain embodiments, the present invention providesmethods for distinguishing between whether to treat a particular tumorin a particular subject with an inhibitor of a kinase, as describedherein, based upon whether or not the tumor has a mutation in a tumorsuppressor gene, such as p53.

In some embodiments, any of the methods further comprise determining alevel of expression or activity of a tumor suppressor gene or a kinasegene (or their encoded gene product) in disease cells, e.g., cancercells, obtained from the subject. In certain embodiments, determiningthe level of expression comprises determining an amount of mRNAexpressed by the tumor suppressor or kinase gene. In certainembodiments, determining the level of expression comprises determiningan amount of polypeptide encoded by the tumor suppressor or kinase gene.In particular embodiments, the kinase gene is listed in FIG. 1C, FIG. 7or FIG. 8. In particular embodiments, the level of expression oractivity of the tumor suppressor or kinase gene (or its encoded geneproduct) is determined prior to providing the subject with theinhibitor. In certain embodiments, the level of expression or activityof the tumor suppressor or kinase gene (or its encoded gene product) isdetermined at about the same time as providing the subject with theinhibitor. In certain embodiments, the level of expression or activityof the tumor suppressor or kinase gene (or its encoded gene product) isdetermined after providing the subject with the inhibitor. In particularembodiments, a subject is treated with the inhibitor if the subject'stumor has a reduced level of expression of the tumor suppressor gene orits encoded protein as compared to the level in a normal control cell,and in other embodiments, the subject is not treated with the inhibitorif the subject has a normal or higher level of expression the tumorsuppressor gene, e.g., p53, as compare to the level in a normal controlcell, e.g., a cell with wild-type p53.

The present invention further provides methods of determining anappropriate treatment or monitoring the therapeutic effect of aninhibitor or treatment, which may be practiced alone or in combinationwith any of the methods described herein.

In one embodiment, the present invention includes a method ofidentifying a subject suitable for treatment with an inhibitor accordingto a method described herein, comprising: (a) determining a level ofexpression or activity of a kinase gene or its encoded gene product indisease cells, e.g., cancer cells, obtained from the subject; and (b)comparing the level of expression or activity of the kinase gene or itsencoded gene product determined in (a) to a control level of expression,wherein a higher level of expression or activity in the disease cells ascompared to the control level of expression or activity indicates thatthe subject is a candidate for the treatment. In particular embodiments,a control level is a level determined in non-disease cells, e.g.,non-disease cells of the same cell type, optionally obtained from thesame subject, or a reference standard, e.g., a level previouslydetermined for non-disease cells. In certain embodiments, the methodfurther comprises providing to the subject an inhibitor of the kinasegene or encoded kinase, if the subject is determined to be a candidatefor the treatment. In particular embodiments, the kinase gene is listedin FIG. 1C, FIG. 7 or FIG. 8. In one embodiment, the kinase gene isWEE1.

In another embodiment, the present invention includes a method ofmonitoring the progression or regression of a disease, e.g., a tumor, inresponse to a treatment, comprising: (a) determining a level ofexpression or activity of a kinase gene or its encoded gene product indisease cells, e.g., cancer cells, obtained from the subject prior to orat about the same time as providing the subject with the inhibitor; and(b) determining a level of expression or activity of a kinase gene orits encoded gene product in disease cells, e.g., cancer cells obtainedfrom the subject following a time period after providing the subjectwith the inhibitor; wherein a reduced level of expression or activitydetermined for (b) as compared to the level of expression of activitydetermined for (a) indicates that the treatment is effective. Inparticular embodiments, the kinase gene is listed in FIG. 1C, FIG. 7 orFIG. 8. In one embodiment, the kinase gene is WEE1.

In certain embodiment, the reduction in kinase activity is determined bycomparing the activity to a reference standard. In a further embodiment,the reference standard is a similar disease cell that is not contactedwith the inhibitor agent. In another embodiment, the reference standardis the same disease cell before it is contacted with the inhibitoragent.

A person of skill in the art will understand that kinase expression oractivity levels, e.g., a reduction in kinase activity or expression, canbe ascertained at the DNA, mRNA, and protein levels. Accordingly, in oneembodiment, the reduction in kinase expression can be determined basedon monitoring the transcriptional activity of the reduction in kinase,i.e., the relative abundance of RNA gene product. For example, commonlyknown methods can by applied to measure abundance of mRNA gene product,such as PCR, quantitative RT PCR. Another method is a nucleaseprotection assay, wherein an antisense probe (labeled with, e.g.,radiolabeled or nonisotopic) hybridizes in solution to an RNA sample.Following hybridization, single-stranded, unhybridized probe and RNA aredegraded by nucleases and intensity of antisense probe is determined fordouble stranded molecules. In yet another embodiment, Northern blotassays are used to detect and ascertain the relative amounts of RNA,such as mRNA, in a sample according to conventional Northernhybridization techniques known in the art.

In additional embodiments, RNA need not be extracted from the diseasecell or control cell. For example, fluorescent in situ hybridization canbe used to determine the presence, relative quantity, and spatialdistribution of target mRNA in a cell. In an illustrative example,Single Molecule RNA FISH (Biosearch Technologies, Novato, Calif.) usesmultiple short singly labeled oligonucleotide probes complementary todistinct portions of the target sequence. When each probe binds to thesingle stranded mRNA template, it causes cooperative unwinding of themRNA, promoting the binding of the additional probes. The net result isthe binding of a large multitude of fluorescent labels to a singlemolecule of mRNA template, providing sufficient fluorescence to reliablylocate each target mRNA in a wide-field fluorescent microscopy image.

Detectable probes, RNA interference molecules and the like useful forany of the methods described herein may be constructed according towell-known techniques based on the sequence of the kinase gene, ornaturally occurring variants thereof.

In another embodiment, the reduction in kinase activity can bedetermined based on monitoring the amount of the polypeptide kinase inthe sample. For example, immunoassays such as Western blot involveimmunoprecipitation of protein from a sample according to methodswell-known in the art. This is followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of the protein sample.After separation of the proteins, immunocytochemistry and the like canby used to determine the amount of the kinase present in the sample. Apreferred agent for detecting a protein of interest is detectableantibody, or fragment thereof, capable of binding to the kinase.

Antibodies can be generated utilizing standard techniques well known tothose of skill in the art. Such antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or an antibody fragment(e.g., Fab or F(ab′)2), can be used. Detectable probes, such asantibodies and the like, useful for any of the methods described hereinmay be constructed according to well-known techniques utilizingpolypeptide moieties containing aspects of the polypeptide sequence ofthe kinase, or naturally occurring variants or derivatives thereof.

Additionally, antibodies, or fragments thereof can be employedhistologically, as in immunofluorescence or immunoelectron microscopy,for in situ detection of kinase protein. In situ detection can beaccomplished by obtaining a histological specimen (e.g., a biopsyspecimen or immobilized cell culture) and applying thereto a labeledantibody that is directed to the kinase polypeptide. The antibody (orfragment) is preferably applied onto a biological sample. Through theuse of such a procedure, it is possible to determine not only thepresence of the protein of interest, but also its distribution withinthe sample. A wide variety of well-known histological methods (such asstaining procedures) can be utilized in order to achieve such in situdetection.

Antibodies can be detected via direct labeling of the antibody via,e.g., coupling (i.e., physically linking) a detectable substance to theantibody, or indirect labeling of the antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody. In some embodiments, the biological sample can bebrought in contact with and immobilized onto a solid phase support orcarrier such as nitrocellulose, or other solid support which is capableof immobilizing cells, cell particles or soluble proteins. The supportcan then be washed with suitable buffers followed by treatment with thedetectably labeled fingerprint gene-specific antibody. The solid phasesupport can then be washed with the buffer a second time to removeunbound antibody. The amount of bound label on solid support can then bedetected by conventional means. A wide variety of known signalingmechanisms are also available for the described immunoassays, such asfluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde fluorescamine, and the like.

In another embodiment, the reduction in kinase activity can bedetermined based on monitoring the enzymatic activity levels of thekinase in a standard kinase assay.

In some embodiments, the composition comprising an inhibitor iseffective to selectively inhibit the growth and/or proliferation of thedisease cells, while not inhibiting the growth and proliferation ofnon-disease cells.

Administration of the composition comprising an inhibitor effective toinhibit the growth and/or proliferation of the disease cells can beperformed according to a variety of well-known methods, which caninclude providing the inhibitor in a pharmaceutical composition, e.g.,comprising a pharmaceutically acceptable diluent, carrier, or excipient.The inhibitor or pharmaceutical composition comprising the inhibitor maybe provided to a cell in vitro (in cell culture), or administered to amammalian subject in vivo by any mode known in the art which retainsinhibitor activity and provides access to the disease cells. Theseinclude, without limitation, oral, parenteral, intravenous,intraperitoneal, subcutaneous, intramuscular, and intrathecal routes ofadministration.

Inhibitors that may be used are described in detail infra. However, incertain embodiments, the inhibitor comprises a small molecule, apolynucleotide, or a polypeptide. In certain embodiments, thepolynucleotide is an antisense RNA, an siRNA, or an miRNA. In certainembodiments, the polypeptide is an antibody or functional fragmentthereof, or an aptamer. In certain embodiments, the inhibitor is a smallmolecule.

Kinase Inhibitors

Inhibitors of any of the kinase therapeutic targets described herein maybe used to practice various embodiments of the present invention, e.g.,to treat or prevent a tumor is a subject. In particular embodiments, akinase inhibitor acts through any of a variety of mechanisms that areeither direct or indirect. Accordingly, kinase inhibitors can inhibitkinases at the DNA, mRNA, or polypeptide levels, targetingtranscription, translation, or functional enzyme (kinase) activity.Kinase inhibitors may be polynucleotides (e.g., DNA or RNA,single-stranded, double-stranded or triple-stranded), polypeptides(e.g., peptides, proteins, or antibodies or fragments thereof), or smallmolecules, or a mixture thereof. These types of inhibitors may beprepared according to methods known and available in the art.

In one embodiment, the inhibitor reduces the expression of a targetkinase, thus reducing the levels of polypeptide product, i.e., thekinase. For example, inhibition of expression can be performed by anagent that physically binds to the DNA encoding the kinase, thuspreventing access to the gene for transcription of the full length mRNA.Inhibition of transcription can also be accomplished, for example, bymodification of the chromatin structure corresponding to the kinase genelocus. In another embodiment, the inhibitory agent binds to or modifiesthe kinase mRNA molecules to prevent translation into the kinasepolypeptide. This can be accomplished, for example, using RNAinterference. In particular embodiments, inhibitors are siRNAs, shRNAsor miRNAs. Such agents that selectively inhibit a target gene may beidentified and prepared using methods known and available in the art,including various publicly available algorithms and software to selectpreferential binding sites on a target gene or mRNA. Sequences ofillustrative siRNAs that may be used according to the methods of thepresent invention are provided in FIGS. 7 and 8.

In another embodiment, the kinase inhibitor inhibits kinase enzymeactivity by binding to the kinase, e.g., the kinase domain, orinterfering with its ability to bind or phosphorylate its substrate. Incertain embodiments, kinase inhibitors are antibodies, or fragmentsthereof, that bind to a target kinase. Antibodies that specifically binda target can be generated utilizing standard techniques well known tothose of skill in the art, e.g., utilizing polypeptide moietiescontaining aspects of the polypeptide sequence of the target kinase.Such antibodies can be polyclonal or monoclonal. An intact antibody, oran antibody fragment (e.g., Fab or F(ab′)2), can be used.

In certain embodiments, a kinase inhibitor is a small molecule.Illustrative, non-limiting examples of such kinase inhibitors includesmall molecules, such as IC261, PF-4800567, and PF-670462. Thestructures of these inhibitors are provided in PCT Publication No.2011/127202. Additional kinase inhibitors include the WEE1 inhibitors,MK-1775 (also referred to as AZD1775) and 681640; the ALK inhibitor,TAE684; the PI3K inhibitor, PI828; the PIK4CB inhibitor, PIK93; the FYNinhibitor, PP2; the FAK inhibitor, PF-562271; and the CHK1 inhibitor,AZD7762; and pharmaceutically acceptable salts thereof. Illustrativestructures of these kinase and other inhibitors or pharmaceuticallyacceptable salts thereof are provided below:

These kinase inhibitors, as well as other kinase inhibitors known in theart, can be used as therapeutic agents, e.g., in the treatment ofcancers with p53 mutations, including head and neck squamous cellcarcinomas, in vivo and in vitro, in accordance with the methodsdescribed herein. In particular embodiments, a kinase inhibitor is usedto treat a cancer where the kinase it inhibits has been identified as atherapeutic target according to a method described herein.

Methods of Identifying Therapeutic Targets and Therapeutic Agents andTreating and Preventing Diseases

As described in the accompanying Examples, the present inventionincludes novel methods of identifying therapeutic targets for variousdiseases. In particular embodiments, these methods utilize one or moreof: (i) high throughput screening to identify therapeutic targets in adisease cell; (ii) target confirmation by high throughput screening ofcandidate therapeutic targets in disease cells of a different species;and (iii) in vitro and/or in vitro functional target validation. Incertain embodiments, these methods are used to identify one or moretherapeutic targets common to a particular type of disease, e.g., aparticular type of cancer. In addition, in particular embodiments, thesemethods are used to identify one or more therapeutic targets for aparticular disease cell in a particular subject, in a form ofpersonalized medicine. Knowledge of therapeutic targets allows theselection of active agents to use to treat the disease. In certainembodiments, the active agents inhibit the expression or activity of atherapeutic target, while in other embodiments, they inhibit theexpression or activity of another gene or protein in the same biologicalpathway as the therapeutic target.

In certain aspects, methods of identifying a therapeutic target for thetreatment or prevention of a disease comprise: (a) screening a pluralityof inhibitory agents, e.g., siRNAs, for their ability to inhibit growthor reduce viability of a disease cell; and (b) identifying one or moregenes targeted by one or more inhibitory agents of (a) that inhibitgrowth or reduce viability of the disease cell. In particularembodiments, the inhibitory agents inhibit the expression or activity ofa gene or its encoded polypeptide. In certain embodiments, methodsfurther comprise the step of obtaining a biological sample comprising adisease or mutant cell from a subject. In particular embodiments, thesubject has been diagnosed with or is considered at risk of developing adisease or mutation.

In one embodiment, a method of identifying a therapeutic drug target ina diseased cell comprises: (a) screening a plurality of inhibitoryagents, e.g., siRNAs, for their ability to inhibit growth or reduceviability of a mammalian diseased cell, or a mammalian cell having adefined mutation, of a first species; (b) identifying one or more genestargeted by one or more inhibitory agents of (a) that inhibit growth orreduce viability of the mammalian cell of the first species; (c)screening a plurality of inhibitory agents, e.g., siRNAs, for theirability to inhibit growth or reduce viability of a mammalian diseasedcell, or a mammalian cell having a defined mutation, of a secondspecies; (d) identifying one or more genes targeted by one or moreinhibitory agents of (c) that inhibit growth or reduce viability of themammalian cell of the second species; and (e) determining one or moregenes identified according to both (b) and (d), wherein the diseasedcell of the first species and the diseased cell of the second speciesshare the same disease, or the mutation in the cell of the first speciesis in the same gene as the mutation in the cell of the second species,and wherein the one or more genes determined according to (e) or theirencoded products are identified as therapeutic drug targets. In certainembodiments, the first species is human, and the second species in anon-human mammal, such as a non-human primate or a mouse.

In certain embodiments, the methods further comprise validating thetherapeutic targets that are identified either in vitro or in vivo,e.g., by determining the ability of an inhibitor of an identifiedtherapeutic target to inhibit growth of other related or unrelateddisease cells. In certain embodiments, this includes determining theability of an inhibitor of an identified therapeutic target to inhibitnon-diseased cell, in order to identify and optionally eliminate targetsthat are not specific to diseased cells, or which have an undesiredeffect on non-diseased cells. In vivo validation methods include, e.g.,the use of animal models of disease, including non-human animalxenograft models of disease, such as non-human animals having humantumor cells grafted to them.

In certain embodiments, the inhibitory agents are screened by contactingthe disease cell or mutated cell with the inhibitory agents, e.g., byinfecting or transfecting cells with the inhibitory agent or a vectorthat expresses the inhibitory agent. In particular embodiment, thescreening is performed using cells in a microarray. In certainembodiments, the effect of the plurality of inhibitors is determined byexamining one or more cell phenotype using a microscope or plate reader.In certain embodiments, the one or more phenotype includes cell growthor proliferation (e.g., growth rate), cell death (e.g., apoptosis), orsecretion of one or more growth factors or cytokines.

Although any types of inhibitors may be used, in particular embodiments,the inhibitory agents are polynucleotides that each selectively inhibitexpression of a target gene. In certain embodiments, the inhibitoryagents are RNA interference agents, siRNAs, shRNAs, or miRNAs. Incertain embodiments, the plurality of inhibitory agents target genes ofa particular class or that encode a particular class of proteins, suchas, e.g., kinases, transcription factors, transcriptional activators,transcriptional repressors, G protein coupled receptors, apoptosisinhibitors, oncogenes, or tumor suppressor genes. In certainembodiments, the inhibitory agents are peptides or proteins, such asantibodies or fragments thereof, or haptens, including those that bindto kinases, transcription factors, transcriptional activators,transcriptional repressors, G protein coupled receptors, apoptosisinhibitors, oncogenes, or tumor suppressor genes, including any of thekinase targets identified herein (see, e.g., FIGS. 1C, 2, 7 and 8). Inother embodiments, the inhibitory agents are small molecules. Candidatecompounds useful in the screening method include compounds from chemicallibraries. Representative useful chemical libraries include libraries ofstructurally diverse compounds, libraries of therapeutic drug-likecompounds, and libraries of therapeutic drugs approved by the Food andDrug Administration (FDA).

In certain embodiments, the disease cell or mutant cell is a tumor cell,such as a primary tumor cell. In particular embodiments, the tumor cellcomprises a mutation in a tumor suppressor gene. In particularembodiments, the tumor suppressor gene is a DNA-PKcs, Atm, p19^(Arf),p53, Hras, Kras, or Prkdc gene. In certain embodiments, the tumor cellhas a mutation in both a p53 tumor suppressor gene and one or more of aHras or Kras gene. In various embodiments, the tumor is a solid tumor ora liquid tumor. In particular embodiments, the tumor is a head or neckcancer, a breast cancer, a prostate cancer, a brain cancer, a thyroidcancer, a lung cancer, an ovarian cancer, a stomach cancer, a pancreaticcancer, a liver cancer, a skin cancer, a leukemia, a lymphoma, a coloncancer, a cervical cancer, a uterine cancer, an esophageal cancer, or abladder cancer.

In related embodiments, the present invention includes methods oftreating or preventing a disease in a subject that involve determiningthe therapeutic agent to use based on the results obtained by a methodsdescribed herein for identifying therapeutic targets. Thus, in certainembodiments, methods for treating or preventing a disease, e.g., acancer, in a subject in need thereof, comprise: (a) screening aplurality of inhibitory agents, e.g., siRNAs, for their ability toinhibit growth or reduce viability of a disease cell obtained from thesubject; (b) identifying one or more genes targeted by one or moreinhibitory agents of (a) that inhibit growth or reduce viability of thedisease cell; and (c) providing to the subject an inhibitor of one ormore of the genes identified in (b). In particular embodiments, themethods further comprises: (d) obtaining a biological sample comprisinga disease cell from the subject.

In other related embodiments, methods for treating or preventing adisease, e.g., a cancer, in a subject in need thereof, comprise: (a)obtaining, ordering or requesting the results of a test comprising: (i)screening a plurality of inhibitory agents, e.g., siRNAs, for theirability to inhibit growth or reduce viability of a disease cell obtainedfrom the subject; and (ii) identifying one or more genes targeted by oneor more inhibitory agents of (i) that inhibit growth or reduce viabilityof the disease cell; and (b) providing to the subject an inhibitor ofone or more of the genes identified according to (a).

In particular embodiments, the disease is a cancer, e.g., a head or neckcancer, a breast cancer, a prostate cancer, a brain cancer, a thyroidcancer, a lung cancer, an ovarian cancer, a stomach cancer, a pancreaticcancer, a liver cancer, a skin cancer, a leukemia, or a lymphoma.

In certain embodiments, the plurality of inhibitory agents targetkinases, transcription factors, transcriptional activators,transcriptional repressors, G protein coupled receptors, apoptosisinhibitors, oncogenes, or tumor suppressor genes.

In particular embodiments, the inhibitor is provided to the subject incombination with another therapeutic agent, e.g., another therapeuticagent used for treatment of the subject's disease.

EXAMPLES Example 1 Identification of Therapeutic Drug Targets for p53Mutant Head and Neck Squamous Cell Carcinoma

Briefly, and as described in more detail below, RNAi kinome viabilityscreens were performed on HNSCC cells including autologous pairs fromprimary tumor and recurrent/metastatic lesions, and in parallel onmurine squamous cell carcinoma (MSCC) cells derived from tumors ofinbred mice bearing germline mutations in Trp53, and p53 regulatorygenes: Atm, Prkdc, and p19^(Arf). Cross-species analysis of cell linesstratified by p53 mutational status and metastatic phenotype wasutilized to select 38 kinase targets. Both primary and secondary RNAivalidation assays were performed on additional HNSCC cell lines tocredential these kinase targets utilizing multiple phenotypic endpoints.Kinase targets were also examined via chemical inhibition utilizing apanel of kinase inhibitors. A preclinical study was conducted on theWEE1 kinase inhibitor, MK-1775.

This functional kinomics approach identified novel survival kinases inHNSCC involved in G2/M cell cycle checkpoint, SFK, PI3K and FAKpathways. RNAi mediated knockdown and chemical inhibition of the WEE1kinase with a specific inhibitor, MK-1775, had a significant effect onboth viability and apoptosis. Sensitivity to the MK-1775 kinaseinhibitor is in part determined by p53 mutational status, and due tounscheduled mitotic entry. MK-1775 displays single-agent activity andpotentiates the efficacy of cisplatin in a p53 mutant HNSCC xenograftmodel.

This study demonstrated that WEE1 kinase is a potential therapeutic drugtarget for HNSCC, and also supports the application of a functionalkinomics strategy to identify novel therapeutic targets for cancer.

Experimental Design

For this study, we hypothesized that HNSCC cancer cells, in particularthose with p53 mutations, are dependent on particular kinases forsurvival and that targeting these kinases could have therapeuticpotential. To identify these cancer-specific survival kinases, weutilized an unbiased and genome scale high-throughput (HT) siRNA genesilencing strategy. We surveyed the entire human kinome to identifythose kinases that are required for survival of HNSCC cells stratifiedby p53 mutational status and metastatic propensity. We included pairs ofHNSCC cells derived from primary tumors and either recurrent ormetastatic lesions. The cell lines derived from the recurrent ormetastatic tumors have been shown by us and others to have moreaggressive features than their primary tumor autologous pairs, asmeasured by migration, avoidance of anoikis, and metastatic potential inmouse orthotopic xenografts (Methods for details) (9).

Recurrent/metastatic tumors are generally resistant to standard of caretherapies and so are most in need of targeted therapies. The rationalefor targeting kinases in human cancer is significant. These enzymesregulate multiple cellular processes that contribute to tumordevelopment and progression, and many human tumors display aberrantactivation of kinases caused by genetic alterations. For tumors that aredependent on kinase activity for survival, targeted drugs could beeffective.

Understanding that human cancer cell lines exhibit genetic andphenotypic heterogeneity, which can hamper the identification of robustdrug targets, we performed a parallel siRNA kinome screen using a set oflow passage murine squamous cell carcinoma (MSCC) cells. These cancercells were derived from tumors of inbred mice bearing germline mutationsin p53 and p53 regulatory genes Atm, Prkdc, and p19^(Arf) (10-13).

This set of p53 pathway deficient cancer cells share the same culturehistory and genetic background and were derived from tumors sharing thesame etiology. The ability to query a set of cells with defects in keyregulators of the p53 response facilitates the nomination of kinasetargets against p53 deficient cancers. Finally, comparative analysis ofsiRNA screen results between mouse and human cells points to survivalkinases that are conserved between species and likely represent the mostrobust drug targets. Through an efficient in vitro and in vivoprioritization and validation scheme, we identified the G2/M cell cycleregulatory kinase WEE1 as one of several clinically promising targets,and show that inhibition of WEE1 with a highly specific small moleculeinhibitor impaired growth of p53 mutant HNSCC tumors in vivo.

Materials and Methods Cell Lines

The following human HNSCC cell lines were used: UM-SCC14A, UM-SCC14C,PCI-15A, PCI-15B, JHU-019, UM-SCC22A, UM-SCC22B; UM-SCC38; UM-SCC17A andUM-SCC47 (5, 14-18). Three cell line pairs were derived from primarytumors and subsequent recurrences or metastatic cervical lymph nodesfrom the same patients: UM-SCC14A, and UM-SCC14C; PCI-15A and PCI-15B;and UM-SCC22A and UM-SCC22B. Cell line JHU-019 was derived from alate-stage OSCC patient (19, 20). The cell lines had the followingcharacteristics.

TABLE 1 Characteristics of HSNCC Cell Lines Metastasis Metastasize toSite of Origin Status of Form Tumor Neck Lymph Node P53 Status and CellLine in Patient Patient in Xenograft in Xenograft Mutation JHU-019Tongue Positive lymph yes yes mutated node frameshift and earlytermination at 169 caused by deletion at 138 PCI-15A* Oral cavityPositive lymph yes/no no mutated node T126Stop PCI-15B* 3^(rd)recurrence Positive lymph yes yes mutated node R273C UM-SCC14A* Floor ofmouth Negative lymph yes no mutated node R280S UM-SCC14C* 3^(rd)recurrence Negative lymph yes n/a mutated node R280S UM-SCC22A*Hypopharynx Positive lymph n/a yes mutated node Y220C UM-SCC22B* Lymphnode Positive lymph n/a yes mutated metastasis node Y220C UM-SCC17ALarynx Negative lymph n/a n/a wild-type node UM-SCC47 Oral cavityPositive lymph n/a n/a wild-type node *Cell line pairs derived from theprimary tumors and subsequent recurrences or metastasis NM = no mutation

For the paired lines, wound-healing assays revealed that the migrationrate of cell lines derived from metastatic HNSCC (i.e. UM-SCC-14C andPCI-15B) was higher than those derived from the primary tumor (i.e.,UM-SCC-14A and PCI-15A) and that JHU-019 had the fastest migration rate(Xu, C. et al., PLoS Genet 2013; 9:e1003169). In addition, JHU-019 andPCI-15B cell lines tested in mouse xenografts by orthotopic injectioninto the tongue produced squamous carcinoma at the sites of injectionand cervical lymph node metastasis (Xu, C. et al., PLoS Genet 2013;9:e1003169).

To determine p53 mutational status, we designed primers to amplify exons2-11 using Primer3 software (Whitehead Institute, Cambridge, Mass.).Primer specificity was confirmed by gel electrophoresis. PCR-amplifiedfragments were purified and sequenced using an ABI 3730xl DNA Analyzerwith ABI's BigDye Terminator Cycle Sequencing method. Sequencing resultsare aligned to GenBank TP53 sequence NG 017013.1 using Sequencher 4.10.1(Gene Codes, Ann Arbor, Mich.). Cell lines were characterized formetastatic potential as described (Xu, C. et al., PLoS Genet 2013;9:e1003169). To determine if a p53 mutation is disruptive, we usedcriteria established by Poeta et al. (Poeta, M L et al., N Engl J Med2007; 357:2552-61).

Murine squamous cell carcinoma (MSCC) cells were derived from NIH/Olastrain mice with germline mutations in p53 pathway genes and included:MSCC-CK101 (Hras^(Q61L) Trp53^(+/+)), MSCC-CK102 (Hras^(Q61L)Trp53^(+/−)), MSCC-CK103 (Hras^(wt) p19Arf^(−/−)), MSCC-CK104(Kras^(G13R) Atm^(−/−)), MSCC-CK1 (Hras^(Q61L) p53^(+/+)) and MSCC-CK4(Hras^(Q61L) p^(53−/− (Cre+p53 lox/lox))) (10-12). MSCC-CK105(Hras^(Q61L) Prkdc^(mu/mu)) cells were from SCID mutant mice of a mixedC3H/Balb/c background (13) The cell lines had the followingcharacteristics.

TABLE 2 Characteristics of MSCC Cell Lines with Germline Mutations inthe p53 Pathway Genotype (Germline Strain P53 Status and Cell LineMutation) Treatment Background Ras status Mutation MSCC-CK101 Wild-typeDMBA/TPA NIH/Ola Hras Q61L Trp53 +/+ (CAA → CTA) Exons 5-8 (NM)MSCC-CK102 Trp53 +/− DMBA/TPA NIH/Ola Hras Q61L Trp53 +/− (CAA → CTA)Exons 5-8 (NM) Δexon 5 (106 bp) MSCC-CK103 P19Arf −/− DMBA/TPA NIH/OlaHras wt Trp53 +/+ Exons 5-8 (NM) MSCC-CK104 Atm −/− DMBA/TPA NIH/OlaKras G13R Trp53 +/+ (GGC → CGC) Exons 5-8 (NM) MSCC-CK105 Prkdc mu/muDMBA/TPA CH3/Balb/cF1 Kras Q61L Trp53 +/+ (CAA → CTA) Exons 5-8 (NM)MSCC-CK1 Wild-type DMBA/TPA NIH/Ola Kras Q61L Trp53 +/+ (Trp53 +/+) (CAA→ CTA) Exons 2-11 (NM) MSCC-CK4 Trp53 −/− DMBA/TPA NIH/Ola Kras Q61L Trp53 −/− (Cre + Trp53 (CAA → CTA) n/a lox/lox) NM = no mutation

All mice were subjected to the identical DMBA/TPA two-stage carcinogenprotocol to induce squamous cell carcinoma. Tumors induced by thisprotocol principally harbor an activating mutation in the Hras oncogene,but mutations in Kras have also been noted. Carcinomas arising from bothp19^(Arf) and p53 deficient mice are highly aggressive and metastatic.Mouse SCC lines were derived using a standard outgrowth explant method.Briefly, carcinoma tissue was washed in sterile PBS, sliced into 2 mmpieces using a sterile razor blade, and placed into a 60 mm tissueculture plate with DMEM media, 10% FCS and Pen/Strep. Media was replacedevery 72 hours until cell outgrowths reached 70-90% confluence, and weresubsequently passaged and/or frozen at low passage number. Total RNA wasisolated from the MSCC-CK1 line with TrIzol and cDNA generated utilizingSuperscript 3 reverse transcriptase (Life Technologies). The p53 cDNAtranscript spanning exons 2-11 was PCR amplified as previously described(Wang et al., Cancer Cell 2009; 16:33-43), and cloned into a TOPO TAvector (Life Technologies), competent cells transformed, and severalcolonies sequenced using an ABI 3730xl DNA Analyzer with ABI's BigDyeTerminator Cycle Sequencing method for mutations in the p53 gene.

High-Throughput RNA Interference Kinome Screens

Kinome-wide siRNA screens were performed with viability as thephenotypic endpoint on five HNSCC lines: JHU-019; PCI15A and 15B;UM-SCC14A and 14C; and five MSCC lines: MSCC-CK101, MSCC-CK102,MSCC-CK103, MSCC-CK104, MSCC-CK105. Normal human foreskin fibroblastswere screened to control for nonspecific cell toxicity. Briefly,culturing of normal human foreskin fibroblasts was performed aspreviously described (Kiyono, T. et al., Nature 1998; 396:84-8).Kinome-wide RNA interference screens were performed on two cultures ofhuman foreskin fibroblasts (HFF1, HFF3) utilizing the Ambion kinomelibrary (Ambion-Life Technologies, Grand Island, N.Y., USA). An HFFexclusion plot was generated using this kinome screen information todetermine whether RNAi mediated knockdown of kinase targets compromisedcell viability in both HFF cultures, with <70% viability (>30% celldeath) as a threshold. siRNA libraries targeting 713 human (MISSION®siRNA Human Gene Family Set, Sigma-Aldrich, St. Louis, Mo., USA; alsoavailable through the Quellos High Throughput Facility at the Universityof Washington Institute for Stem Cell and Regenerative Medicine,Seattle, Wash. USA) and 572 murine kinases (Ambion-Life Technologies)were constructed and utilized in pools of 3 independent siRNAs targetingeach gene, in a one gene per well approach. RNAi screens were performedin 384-well format utilizing robotics instrumentation (Toyoshima, M. etal., Proc Natl Acad Sci USA 2012; 109:9545-50). Transfection feasibilityof each cell line was established using a factorial optimization. Mockcondition and a non-targeting universal siRNA control were utilized asnegative controls, while a siRNA directed at KIF11 (kinesin-likeprotein), which arrests cells in mitosis was utilized as a positivecontrol. All reagent conditions were statistically evaluated using asimple Z-factor score to evaluate differentials and variability ofreplicates (i.e. potent cell killing with KIF11 at the lowest toxicitypossible in the mock universal controls) to select an optimizedtransfection condition for each cell line (Zhang, J H et al., J BiomolScreen 1999; 4:67-73). All kinases were tested in triplicate toestablish experimental variability and statistical validity. ScrambledsiRNA negative controls were used to monitor dynamic range andoff-target effects and the results were standardized to mock-transfectedcells. Viability and apoptosis were quantified utilizing an EnvisionMultilabel detector/plate reader (Perkin Elmer) with either aCellTiterGlo assay (Promega), or Apotox assay (Promega), the formermeasures metabolic ATP via a standard curve to mock/universal siRNA atall conditions. Raw luminescence values were then Z transformed per cellline and plotted for distribution and data mining (Miner 3D software,version 7.3). All HT kinome screens and subsequent validation screens onMSCC and HNSCC cells were statistically evaluated using publishedmethods (Birmingham, A. et al., Nat Methods 2009; 6:569-75).

Comparison of Human and Mouse Kinome Screens

The 713 human and 572 murine kinase sets were cross-referenced utilizingmouse genome informatics (MGI) (wwwdotinformaticsdotjaxdot.org/) andNational Center for Biotechnology Information (NCBI)(wwwdotncbidotnlmdotnihdotgov/) database nomenclature to generate acommon list of 508 kinases referred to as the interspecies kinome. Usingthis common list, prioritization of screen results then followed basedon the viability scores from the 5 HNSCC cells and 5 MSCC cell lines.Mean viabilities (μ_(i2)) from 5 HNSCC cell lines [μ_(i2) All(human)=μ_(i) (019)+μ_(i) (14A)+μ_(i) (14C)+μ_(i) (15A)+μ_(i) (15B)] andfrom 5 MSCC cell lines were calculated [μ_(i2) All (murine)=μ_(i)(WT)+μ_(i) (Trp53+/−)+μ_(i) (p19^(Arf)−/−)+μ_(i) (Atm−/−)+μ_(i) (Prkdcmu/mu)] for each of 508 kinases {i=1, 2, 3, 4 . . . 508}, where μ_(i) isthe triplicate of pooled siRNAs (3 distinct siRNAs) average normalizedviability for each individual gene per cell line. Mean viabilities(μ_(i2)) for each gene were then Z transformed using the equation,Z=μ_(i2)−μ/σ where μ is the mean viability and σ the standard deviationfor all siRNAs/wells for all 5 HNSCC cell lines and all 5 MSCC celllines, respectively. Mean viabilities (μ_(i2 All), μ_(i3 p53 mutant),μ_(i4 metastatic)) and Z score transformations were calculated for bothhuman and murine lines per genotype and phenotype for: (1) all human andmurine cell lines; (2) p53 mutant/deficient human and murine cell lines;and (3) metastatic human and murine cell lines. Cartesian plots(Z-score_(murine), Z-score_(human)) of all 508 kinases in common withmurine and human kinomes were then generated for each of the threecomparisons. Population mean viabilities (Mean_(All),Mean_(p53 mutant All), Mean_(Metastatic)) and Z-score transformations(Z-score_(Mean All), Z-score_(p53 mutant All), Z-score_(Mean Met)) werecalculated for specific genotypic and phenotypic comparisons andutilized in the color-coded overlay on the cartesian plots for selectionper comparison. Kinase targets were selected based on Z-score thresholdfrom each of the cartesian plots: Z-score_(Mean All)<−1.0;Z-score_(p53 mutant All)<−2.0, Z-score_(Mean Met)<−1.5, where kinaseswere data mined from more then one comparison and duplicates wereremoved for a final selection of 38 kinase targets from all threecomparisons, kinase targets (38 kinases)=kinase targets_(All)+kinasetargets_(p53mutant)+kinase targets_(Met) (FIGS. 1B and 1C). Thesekinases included serine/threonine kinase 32B (STK32B); aurora kinase A(AURKA); galactokinase 2 (GALK2); thymidine kinase 2 (TK2),mitochondrial NUAK family, SNF1-like kinase 2 (NUAK2);calcium/calmodulin dependent protein kinase II beta (CAMK2B); membraneprotein, palmitoylated 3 (MAGUK) (MPP3); serine/arginine rich proteinspecific kinase 3 (STK23); phosphatidylinositol-4-phosphate 5-kinase,type 1, beta (PIP5K1B); glycerol kinase 2 (GK2); ribosomal protein S6kinase-like 1 (RPS6KL1); phosphatidylinositol 4-kinase, catalytic, beta(PIK4CB); FYN oncogene related to SRC, FGR, YES (FYN); eukaryotictranslation-initiation factor 2 alpha kinase 3 (EIF2AK3); inhibitor ofkappa light polypeptide gene enhancer in B cells, kinase epsilon(IKBKE); Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog(FGR); mitogen-activated protein kinase kinase kinase 8 (MAP3K8);uridine-cytidine kinase 1-like 1 (UCKL1); tribbles homolog 2(drosophila) (TRIB2); eukaryotic translation-initiation factor 2 alphakinase 4 (EIF2AK4); TXK tyrosine kinase (TXK); EPH receptor 3 (EPHA3);anti-Mullerian hormone receptor, type II (AMHR2); Wee1 homolog (S.pombe) (WEE1); Chk1 checkpoint homolog (S. pombe) (CHK1);mitogen-activated protein kinase 13 (MAPK13); TTK protein kinase (TTK);fms-related tyrosine kinase 1 (FLT1); phosphoinositide-3-kinase,catalytic, beta polypeptide (PIK3CB); Integrin-linked kinase (ILK);anaplastic lymphoma receptor tyrosine kinase (ALK); NIMA (never inmitosis gene a)-related expressed kinase 4 (NEK4); WNK lysine deficientprotein kinase 4 (WNK4); c-abl oncogene 1, non-receptor tyrosine kinase(ABL1); v-erb-b2 erythroblastic leukemia viral oncogene homolog 3(avian) (ERBB3); mitogen-activated protein kinase kinase kinase 2(MAP3K2); fibroblast growth factor receptor 3 (FGFR3); and Proteinkinase C, Epsilon (PKCE). Kinases were further prioritized based onthose whose expression or activity was increased in squamous cellcarcinoma (SCC) and that play known functions in SCC pathogenesis.

Primary Validation of Kinase Targets with siRNA

Twenty-eight kinases were selected for follow-up and validation based onthe interspecies kinome comparison and HFF kinase exclusion, includingABL1, CANK2B, EIF2AK3, EIF2AK4, EPHA2, FGFR3, FGR, FYN, GALK2, IKBKE,ILK, MAP3K2, MAP3K8, MAPK13, MPP3, NEK4, NUAK2, PI4 KB, PRKCE, RPS6KL1,STK32B, TK2, TRIB2, TTK, TXK, UCKL1, WEE1, and WNK4. Two small-scaletime-course primary validation RNAi screens were performed in parallelon four HNSCC cell lines (UM-SCC14A, UM-SCC14C, PCI-15A, PCI-15B) in384-well formats with an independent set of siRNAs (Qiagen, FIG. 7),with three separate siRNAs plus pooled siRNAs per gene target intriplicate for an N=12 for each gene target. All phenotypic endpoints ofcell viability and caspase 3/7-dependent apoptosis were measured inparallel screens in a time-course format at 1.5, 3, and 4.5 dayspost-transfection using the CellTiter-Glo assay (Promega) and Apotox-Glo(Caspase-Glo 3/7 reagent) assay (Promega) per manufacturersspecifications and an Envision multilabel plate reader (PerkinElmer).Primary screen “hits” were assessed utilizing a single endpoint for cellviability at 4.5 days post-transfection termed, absolute viability, byboth a negative-control independent analysis: triplicate siRNAs versuspopulation mean of the screen, Z-score threshold and unpaired t-test,Z-score <−1.0, P<0.1 scored as hit, as well as a negative-controldependent analysis: triplicate siRNAs versus universal negative controlsiRNAs, ANOVA with Dunnetts post-test, mean difference >0 and P<0.05scored as hit. Comparing post-transfection effects on absolute viability(day 4.5) for each kinase target with either the universal negativecontrol siRNA or the population mean yielded similar results.Differential viabilities (day 4.5-1.5) based on the mean of both theN=12 data (i.e., all three distinct siRNAs per target plus pooled siRNAsin triplicate) and N=3 data (i.e., pooled siRNAs alone) were calculatedfor all 28 kinase targets listed in FIG. 7. AUC (area under the curve)estimates of caspase-dependent apoptosis (AUC_(estimate)) utilizing allthree data points with both the N=12 data and N=3 pooled siRNAs werecalculated for all kinase targets. Statistical significance of RNAinterference mediated knockdown of the 28 kinases was assessed via ANOVAwith Dunnetts post-test for multiple comparisons (P<0.05 as significant)on differential viability (day 4.5−day 1.5), and area under the curve(AUC) analysis of caspase 3/7 dependent apoptosis versus universalnegative siRNA control (UNI).

Secondary Validation of Kinase Targets with siRNA

Ten kinase targets were further validated in a 96 well format in fiveadditional HNSCC cell lines (UM-SCC22A, UM-SCC22B, UM-SCC38, UM-SCC47A,JHU-019). In addition, the ten HFF exclusion kinase targets wereincluded in the low-throughput assay (i.e., 20 kinase targets listed inFIG. 8). This assay consisted of three independent siRNAs per well(pooled siRNAs) assayed in triplicate (Qiagen, FIG. 8) for cellviability and apoptosis measured at 1.5, 3, and 4.5 dayspost-transfection utilizing the Apotox-Glo assay (Promega, Madison,Wis.) as per manufacturers specifications utilizing a Synergy H4 HybridMulti-Mode microplate reader (Biotek). Statistical significance of RNAinterference mediated knockdown of the 20 kinases was assessed via ANOVAwith Dunnetts post-test for multiple comparisons on absolute viability(day 4.5), differential viability (day 4.5−day 1.5), and area under thecurve (AUC) analysis of caspase dependent apoptosis versus the negativesiRNA control (SINC).

Dose-Response Curves with Kinase Inhibitors

Kinase inhibition dose response curves were performed with six kinaseinhibitors (MK-1775 (a.k.a., AZD-1774), TAE684, PI828, PIK93, PP2,PF-562271) against kinase targets (WEE1, ALK, PI3K, PIK4CB, FYN, FAK(ILK surrogate)), respectively. Kinase inhibitors: MK-1775 (S1525),PIK93 (S1489), TAE684 (S1108) were obtained from Selleck Chemicals(Houston, Tex.); PI828 (2814), PP2 (1407) from Tocris Bioscience (Mpls,MN), and PF-562271 from Synkinase (San Diego, Calif.). All HNSCC(UMSCC-17A, UM-SCC47A, PCI-15A, PCI-15B, UM-SCC14A, UM-SCC14C) and MSCC(CK1: p53+/+ and CK4: p53−/−) cells were plated at −5-10×10³ cells/100μl per well, and incubated at 37° C. for ˜24 hours on 96-well assayplates (Corning Inc.). Serial dilutions of the kinase inhibitors andvehicle control (DMSO) were prepared in 1 mL assay blocks at 3× workingconcentration to generate dose-response curves ranging from 100 μM to0.03 μM. All serial dilutions were prepared using cell culture media.Approximately 72 hours post-treatment, cells were assessed for metabolicactivity via ATP using CellTiterGlo (Promega), following the protocoloutlined by the manufacturer utilizing an FLx800, and/or a Synergy H4Hybrid multi-mode reader (Biotek). All assays were performed intriplicate and normalized to wells with no treatment. Dose-responsecurves and IC₅₀ values were generated using GraphPad Prism Version 5(Parameters: nonlinear regression fit; equation=log (inhibitor) vs.response−variable slope (four parameters); single constraint).

COSMIC Public Database of Drug Sensitivity Data

Utilizing the catalogue of somatic mutations in cancer (COSMIC) website,TP53 gene mutational status was extracted from the Sanger Cancer CellLine Project which contains information on 820 cancer cell lines. Inaddition, the Genomics of Drug Sensitivity Project (Release 2 Jul. 2012)contains 541 cancer cell lines that were treated with a WEE1/CHK1inhibitor, 681640 (EMD Millipore), a pyrrolocarbazole compound that actsas a potent, ATP-binding site inhibitor of WEE1 (IC₅₀=11 nM). Drugsensitivity was measured with 9 different concentrations of 681640 andhalf maximal inhibitory (50%) values presented as natural log (μM). Thesign test was applied to test the median difference in sensitivity byTP53 status. We performed a similar analysis on only the squamous cellcarcinoma (SCC) cell lines. Forty-two squamous cell lines wereidentified from COSMIC annotation, eight were p53 wild type and theremaining 34 had a p53 mutation.

Mitotic Entry, Cell Cycle Analysis, Apoptotic Assays

Mitotic entry was assessed as previously described (Aarts, M. et al,Cancer Discov 2012; 2:524-39). Briefly, HNSCC cells (PCI-15B, UMSCC-17A)were treated with 1 μM MK-1775 for 8 and 24 hours, and all cells wereharvested, washed, and incubated with rabbit monoclonal antibody tophospho-histone H3 (Serine10) (Cell Signaling Technology, Cat. No. 3465,Danvers, Mass.) for 2 hours at RT, washed, and DNA stained with 20 μg/mlpropidium iodide, RNaseA in PBS (Sigma-Aldrich; Cat. Nos. P4170, R6513).Flow cytometric analysis was performed utilizing a BD FACS Canto II, andprofiles analyzed with BD Cell Quest software (Becton Dickinson, CA).Caspase-3/7 dependent apoptosis was assessed as per protocol (Promega,Cat. No. G8091) utilizing a SynergyH4 Hybrid Reader (Biotek, VT). AUCswere calculated for all treatments and vehicle (DMSO) for all cell linesutilizing two measurements over a 48-hour period with threeconcentrations (30 nm, 100 nm, 1 μM) of MK-1775.

Xenograft Tumor Model

PCI-15B cells were inoculated subcutaneously into the right flanks of 28eight-week old NOD/SCID IL2 gamma null mice (NSG) provided by the Olsonlaboratory at the Fred Hutchinson Cancer Research Center. When tumorsreached a palpable mass of >50 mm³, mice were randomly assigned intofour treatment groups of seven mice each and all measurements andtreatment regimens were carried out using a double-blind protocol. TheWEE1 inhibitor MK-1775 (S1525-Selleck Chemicals, Houston, Tex.) wasdelivered by oral gavage (30 mg/kg) twice per week for 4 weeks indimethylsulfoxide in 0.5% methylcellulose (M0512, Sigma Aldrich) in a1:14 suspension. Cisplatin (P4394, cis-diammineplatinum (II) dichloride,Sigma Aldrich) was delivered via intra-peritoneal injection (4.0 mg/kg)once a week for 4 weeks. The volume of the implanted tumor was measuredweekly with calipers and tumor volumes calculated using the formula:V=L×W²/2; in which V, volume (mm³); L, largest diameter (mm); W,smallest diameter (mm). All animal protocols were approved by the FredHutchinson Cancer Research Center Laboratory Animal Care and UseCommittee.

Immunoblotting

Tumor tissues were minced and homogenized on ice in M-PER MammalianProtein Extraction Reagent supplemented with Halt Protease andPhosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Extractedproteins were quantified by a BCA protein assay (Thermo FisherScientific). Fifty μg of each protein specimen was revealed on a NuPAGE®4-12% Bis-Tris mini gel (Life Technologies) and transferred onto anImmobilon-P PVDF membrane (Millipore, Billerica, Mass.). Anti-WEE1 (Cat.No. 4936), anti-phospho-WEE1 (Ser642) (Cat. No. 4910), anti-CDC2 (Cat.No. 9112), and anti-phospho-CDC2 (Tyr15) (Cat. No. 4539) antibodies werepurchased from Cell Signaling Technology (Danvers, Mass.). The secondaryantibodies used were ZyMax™ horseradish peroxidase (HRP) conjugatedgoat-anti-rabbit IgG (Life Technologies). HRP was detected withSuperSignal West Pico Chemiluminescent Substrate kit (Thermo FisherScientific).

Statistical Analysis

All column and curve data points presented as mean+/−sem, unlessotherwise noted. All statistical analyses were performed using unpairedtwo-tailed t-tests unless otherwise indicated. All statistical analysisof RNAi interference primary and secondary screening data is describedabove and all calculations used for significance testing are presentedin supplemental tables. Statistical tests were all performed utilizingGraphpad Prism versions 5 & 6 (GraphPad Software Inc., CA).

Results RNA Interference Kinome Screens of Squamous Cell Carcinoma

We performed kinome wide siRNA viability screens on a set of five HNSCCcell lines (UM-SCC14A; UM-SCC14C; PCI-15A; PCI-15B and JHU-019). Twopairs of these cells (UM-SCC14A UM-SCC14C and PCI-15A, PCI-15B) werederived from primary and subsequent post-treatment recurrences ormetastatic cervical lymph nodes from the same patients and all carrieddisruptive mutations in p53. A total of 713 kinases were interrogatedusing an arrayed siRNA platform that quantified cell viability followingknockdown with a pool of 3 siRNAs/gene/well. All assays were performedin triplicate. Cell viability was monitored 4 days after siRNAtransfection using the ATP-based, CellTiterGlo assay (see Methods fordetails). In parallel, we performed kinome-focused screens on a set offive low passage cancer cells derived from murine squamous cellcarcinomas (MSCC). These cells were isolated from carcinoma bearinginbred mice harboring germline mutations in the p53 pathway genes Atm,Prkdc, p19^(Arf), and Trp53.

Next, we derived an interspecies kinome (508 kinases shared between bothspecies) to identify kinases in which RNA interference mediatedknockdown negatively impacted cell viability in both human and mousecells (see schematic in FIG. 1A). Cell screens were further stratifiedby p53 mutant status and metastatic propensity. Cartesian plots withcross-species comparison of viability Z scores for all 508 kinases, forall cell lines (left), p53 mutant cell lines (middle), and metastaticcell lines (right) are shown in FIG. 1B. Thirty-eight kinases wereselected for follow up based on several criteria, including sharednegative Z scores in both species and specificity to cells with mutantp53 and metastatic phenotype (FIG. 1C). Many of these putative HNSCCsurvival kinases are implicated in signaling pathways such as focaladhesion and integrin signaling (CAMK2B, FYN, ILK, EPHA3, EIF2AK4,TRIB2), PI3K signaling (PIK4CB, PIK3CB, PIP5K1B, TRIB2, FGFR3, ALK), SRCsignaling (FYN, TXK, CAM2 KB), and G2/M cell cycle regulation (WEE1,NEK4, TTK, AURKA, CHK1).

In order to prioritize targets for preclinical validation, we usedprimary cultures of human foreskin fibroblasts (HFF) to assess whetherinhibition of these kinases caused toxicity to normal cells. Ten kinasescaused >30% loss in cell viability in both HFF cultures (FIG. 10) andwere not included in the primary validation screen. The remaining 28kinase targets were retested with independent siRNAs on the same twopairs of autologous HNSCC cell lines (UM-SCC14A and 14C; PCI-15A and15B) using a format of three separate siRNAs per gene plus a pool of allthree siRNAs, each in triplicate (i.e., N=12 per gene) (FIG. 9). Bothcell viability and caspase 3/7-dependent apoptosis were measured inparallel at 1.5, 3, and 4.5 days post-transfection. Differentialviability (day 4.5-1.5), absolute viability (day 4.5), and apoptosis wascalculated for each kinase (see Methods for details). Differentialviabilities were calculated in order to measure post-transfectioneffects over time (day 4.5-1.5) and statistically evaluated versus theuniversal negative control siRNA (FIG. 2A). Differential viabilitiescalculated utilizing three separate siRNAs per gene plus the pool of allthree siRNAs yielded comparable results.

We focused on the pooled analysis to reduce off-target effects (31).Likewise, results obtained utilizing either differential or absoluteviability metrics yielded similar prioritized kinase targets (seeMethods for details). To measure the cumulative effects of RNAi mediatedknock-down on apoptosis, area under the curve estimates ofcaspase-dependent apoptosis was determined versus the universal negativecontrol siRNA (FIG. 9 and FIG. 2B). Results from this primary validationscreen revealed that many of the kinase targets that significantlyreduced viability also increased apoptosis (FIG. 2A). RNAi mediatedknockdown of NEK4 and WEE1 kinases led to a significant reduction incell viability in all four cell lines tested, while targeting TRIB2 didso for three of the four lines.

Ten kinase targets (NEK4, WEE1, ILK, CAM2 KB, FGFR3, FYN, PI4 KB(PIK4CB), TRIB2, TTK, TXK) that caused a significant reduction inviability and/or increase in apoptosis following siRNA transfection inat least one cell line were selected for secondary validation on fiveadditional HNSCC lines (UM-SCC22A, UM-SCC22B, UM-SCC17, UM-SCC47 andUM-SCC38), and kinase target metrics were measured and calculated usingthe same format as above (FIG. 9). UM-SCC22A and UM-SCC22B are anautologous pair of cells derived from a primary tumor and cervical lymphnode metastasis from the same patient. We also retested ten kinases fromthe discovery screen whose siRNAs reduced viability in HFFs (FIG. 2B andFIG. 9).

RNAi mediated knockdown of kinases that regulate the G2/M transition,NEK4, WEE1, AURKA, and CHK1, as well as FYN and CAM2 KB significantlyimpaired viability in three or more of the five cell lines tested (FIG.2B) while knockdown of the WEE1, NEK4, and AURKA kinases induced thehighest levels of apoptosis in all five HNSCC cell lines. Altogether,compiled primary and secondary validation data showed that RNAinterference mediated knockdown of WEE1, NEK4, and AURKA kinasessignificantly reduced viability and increased apoptosis in over 75% ofHNSCC cell lines (FIG. 2C).

A comparison of functional kinomic profiles between cell line pairsisolated from the same patients showed a high degree of concordance, butwith a tendency for the metastatic/recurrent cells to be more resistantto kinase knockdown relative to their primary tumor cell pair (FIG. 2and FIG. 11). For example, RNAi mediated knockdown of TK2 and TRIB2 wasmore effective in cells isolated from the primary vs. therecurrent/metastatic lesions (FIG. 2A and FIG. 11). Several kinases,such as WEE1 and NEK4, remained equally effective in both primary andrecurrent/metastatic tumors.

Small Molecule Inhibition of Kinases Confirms Role in HNSCC CellSurvival

We further prioritized kinase targets using commercially available smallmolecule inhibitors. This step not only provides independent chemicalconfirmation of siRNA results but also provides lead compounds to testin in vivo models. Dose-response curves utilizing kinase inhibitors wereperformed for WEE1 (MK-1775), ALK (TAE684), PIK4CB (PIK93), FAK(PF-562271), PIK3CB (PI828), and FYN (PP2) (FIG. 3). In agreement withour siRNA knockdown experiments, p53 mutant HNSCC cell lines weresensitive to small molecule inhibitors targeting WEE1, ALK, PIK4CB, andFAK. The WEE1 kinase inhibitor, MK-1775 had the broadest and mostsignificant effect on cell survival in both primary andrecurrent/metastasis derived HNSCC cells, with an IC₅₀ ranging from 220nM-3.1 μM (FIG. 3).

Preclinical Validation of WEE1 as a Drug Target for HNSCC In Vitro andIn Vivo

Results from our cross-species comparative analysis of kinome screens,validation assays, and small molecule inhibitor studies nominated WEE1,a G2/M regulator, as a promising target against p53 mutant HNSCC. Tofurther explore the sensitivity of p53 deficient cells to WEE1inhibition, we performed dose-response curves with MK-1775 in pairs ofp53 wild type and p53 mutant/deficient SCC cells. The IC₅₀ for MK-1775was 20-fold lower in p53−/− MSCC cells compared to p53 wild type cells(0.22 μM vs. 4.5 μM) (FIG. 4A). The IC₅₀ for MK-1775 in p53 mutantPCI-15A and PCI-15B HNSCC cells (0.14-0.17 μM) and p53 wild typeUMSCC-17A cells (4.5 μM) showed a similar differential sensitivity toMK-1775 as the mouse SCC cells, while p53 mutant UMSCC-14A and UMSCC-14Ccells and p53 wild type UMSCC-47 cells showed intermediate IC₅₀ valuesof 0.58-1.10 μM (FIG. 4B).

As WEE1 regulates mitotic entry, this suggests p53 deficient cells aresensitive to deregulation of the G2/M transition. CHK1, a kinaserequired for the DNA damage induced G2/M checkpoint, and AURKA, a kinaseinvolved in spindle assembly during mitosis, were also identified asputative survival kinases (FIG. 2). Consistent with the WEE1 inhibitorresults, p53 deficient MSCC cells were also more sensitive to the CHK1inhibitor, AZD7762 than p53 wild type cells (IC₅₀ 0.13 μM vs. 2.2 μM)(FIG. 4A).

Data from the Genomics of Drug Sensitivity project containsdose-response measurements on 820 genomically characterized cancer celllines treated with 138 different compounds (32). One compound in thiscollection, labeled 681640, is a dual WEE1/CHK1 inhibitor (33). We usedmutational profiling data from the Sanger Cancer Cell Line Project toclassify cell lines based on p53 mutational status and tested forcorrelation with sensitivity to 681640 (34). Examination of the 42 cellsthat were derived from squamous cell carcinomas of the head and neck(19), oesophagus (11), lung (5), cervix (3), vulva (3), and skin (1),showed that, despite a broad range of sensitivities in both wild-typeand mutant groups, on average p53 mutant SCC cells had increasedsensitivity to 681640 compared to p53 wild type cells (median IC₅₀:5.34μM vs. 29.23 μM, P=0.005) (FIG. 4C). However, the correlation betweenp53 status and sensitivity to 681640 was not observed in the overallcollection of 499 cell lines, which excluded the 42 SCC cell lines andrepresents a broader variety of tumor types. In fact, the trend betweenp53 mutant status and 681640 sensitivity was reversed (median IC₅₀:12.83 μM vs. 7.75 μM P=1.348×10⁻⁵) (FIG. 4C). This analysis emphasizesthe importance of validating candidate synthetic lethal interactions ordrug sensitivities in specific tumor contexts and it indicates thatother factors besides p53 mutational status affect sensitivity to681640.

We next used flow cytometric cell cycle analysis to determine the basisfor the enhanced sensitivity of p53 mutant SCC cells to WEE1 inhibition.Treatment of cells with MK-1775 led to unscheduled mitotic entry in p53mutant but not wild type cells as measured by phospho-histone H3 (serine10) (FIG. 5A). This was accompanied by an increase in subG1 DNA content,a loss of 4N DNA content, and activation of the apoptotic marker,caspase 3/7 (FIGS. 5B and 5C). This indicates that WEE1 inhibition byMK-1775 in p53 mutant SCC cells caused unscheduled mitotic entry leadingto mitotic catastrophe and apoptotic cell death.

To determine if WEE1 inhibition was effective against p53 mutant HNSCCin a preclinical tumor model, we performed a four arm double-blind studyon PCI-15B xenograft bearing mice. When tumors reached a palpable massof >50 mm³, mice were randomized into four treatment arms and treatedwith vehicle, MK-1775, cisplatin, or cisplatin plus MK-1775. Cisplatinis the standard chemotherapeutic agent for HNSCC and cisplatin plusMK-1775 was used to determine if inhibition of WEE1 would synergize withDNA damaging therapy, as p53 mutant tumor cells would be expected todepend on G2/M arrest after DNA damaging treatment to repair DNA. Twiceweekly oral gavage of MK-1775 inhibited growth of HNSCC tumors by 66%over the 4-week protocol as compared to vehicle (FIGS. 6A and 6B;P=0.06). Cisplatin alone led to partial tumor regression, but alsocaused significant weight loss (FIG. 12). However, MK-1775 given 24hours after cisplatin therapy further augmented tumor regression (60%reduction with cisplatin alone vs. 80% reduction with cisplatin plusMK-1775, P=0.003). Tumor lysates from MK-1775 treated mice probed withWEE1 and CDC2 antibodies showed reduced phosphorylation of WEE1 and itssubstrate CDC2 indicating that oral administration of MK-1775effectively blocked WEE1 kinase activity in tumors (FIG. 6C).

Discussion

One of the most significant clinical challenges in the management ofHNSCC patients is recurrent disease. In addition to being resistant toradio or chemotherapy, these tumors can present with distant metastases,leaving palliative care as the only option. Here we applied a functionalkinomic approach to identify new candidate therapeutic targets foraggressive p53 mutant tumors. To prioritize targets we also screenedmurine SCC cells, which are defective in the p53 response and have ahigh propensity to metastasize. We focused on kinase targets that wereeffective in both species and in more aggressive p53 mutant cells.Retesting of these prioritized targets with independent siRNAs usingboth viability and apoptosis endpoints pinpointed those that wereeffective in most or all cells tested, as well as those that were cellline or condition specific.

Comparing siRNA kinome screening results from cells derived from primaryand recurrent/metastatic lesions revealed a high degree of concordance,implying that tumor cells isolated at different times or locations fromthe same patient share common vulnerabilities. In addition,recurrent/metastatic cell lines tended to be less responsive to kinaseknockdown relative to cells from the primary lesion, suggestingdevelopment of resistance to target knockdown induced cell death.Altogether, these findings indicate this functional kinomic platform canreliably identify profiles of essential survival kinases specific toindividual patients.

To further validate candidate therapeutic targets we tested severalsmall molecule kinase inhibitors as a confirmatory step to support theRNAi results as well as to identify those inhibitors that might beeffective for testing in vivo. Overall, this strategy identified theWEE1 kinase for further validation in vivo as RNAi mediated knockdown ofWEE1 led to a significant reduction in cell viability and a concomitantincrease in apoptosis in all nine HNSCC cell lines tested. Moreover, p53deficient MSCC and HNSCC cells were highly sensitive to the specificWEE1 inhibitor MK-1775 relative to p53 WT cells, a finding that has beenobserved in other settings (Hirai, H. et al., Mol Cancer Ther 2009;8:2992-3000; Bridges, K A et al., Clin Cancer Res 2011; 17:5638-48; andRajeshkumar, N V et al., Clin Cancer Res 2011; 17:2799-806). Furthermore, the HPV+p53 wild-type cell lines were more sensitive to MK-1775than the p53 wild-type cells, consistent with the idea than functionalloss of p53, either by E6 viral component of HPV or by somatic mutationis associated with greater sensitivity to the WEE1 inhibitor, MK1775(FIG. 4D). Mechanistically, WEE1 inhibition in p53 mutant cells, but notWT cells, led to unscheduled mitotic entry, mitotic catastrophe, andapoptosis, consistent with previous reports (Aarts, A. et al., CancerDiscov 2012; 2:524-39; and De Witt Hamer, P C et al., Clin Cancer Res2011; 17:4200-7). The G2 checkpoint kinase CHK1 was also a top candidatefrom our screen and p53 deficient SCC cells showed an increasedsensitivity to both a CHK1 and a dual WEE1/CHK1 inhibitor. Collectivelythis suggests p53 deficient SCC cells may be particularly vulnerable toderegulation of the G2/M transition.

As preclinical validation, we demonstrated that oral administration ofMK-1775 inhibited the growth of p53 mutant HNSCC xenografts, and alsocooperated with cisplatin to induce tumor regression. Our comparablyconservative MK-1775 dosing regiment of two times per week, 24 hourspre- and post-cisplatin treatment was performed in the context oflimiting potential toxicities from cisplatin treatment. This regime waswell-tolerated, suggesting that higher doses of the MK-1775 inhibitorwould also be well-tolerated and high efficacy could be obtained eitheras a single-agent or in combination with genotoxic treatment.

Currently, cisplatin chemotherapy for the treatment of HNSCC is giveneither in the neoadjuvant setting or concurrently with radiotherapy.Unfortunately, the associated toxicities of combining cisplatin withother chemotherapeutic agents or the three potentially toxic cisplatindoses of 100 mg/m2 administered during radiation treatment can limit theclinical applicability of these regimens. Thus, the degree by whichMK-1775 enhances response to cisplating would not only increase theeffectiveness of existing therapy, but would open the possibility ofreducing cisplatin dosing to minimize side effects and broaden patientcandidacy to these regimens.

MK-1775 has been shown to sensitize other p53 mutant tumors to DNAdamaging agents (Hirai, H. et al., Mol Cancer Ther 2009; 8:2992-3000;Bridges, K A et al., Clin Cancer Res 2011; 17:5638-48; and Rajeshkumar,N V et al., Clin Cancer Res 2011; 17:2799-806). Molecular analysis ofHNSCC tumor lysates showed reduced phosphorylation of the WEE1 substrateCDC2, indicating that MK-1775 inhibited its intended target. Wepreviously found amplification of 11q13.1 in metastatic HNSCC tumorcells with corresponding over-expression of cyclin B, the activatingsubunit of CDC2 (Xu, C. et al., Mol Cancer 2010; 9:143), which couldexacerbate the sensitivity of HNSCC cells to WEE1 inhibition.

In addition to WEE1 and CHK1, siRNAs to other mitotic kinases includingAURKA and NEK4 reduced viability and increased apoptosis in the majorityof HNSCC cells including those derived from recurrent/metastatic lesionssuggesting potential as therapeutic targets. AURKA and CHK1 are beingpursued as drug targets (Dar, A A et al., Mol Cancer Ther 2010;9:268-78; Katayama, H. et al., Biochim Biophs Acta 2010; 1799:829-39;and Ma, C X et al., Trends Mol Med 2011; 17:88-96), while NEK4 a memberof the NIMA family of kinases modulates sensitivity to microtubulepoisons and DNA damage (Doles, J. et al., Cancer Res 2010; 70:1033-41;Moniz, L. et al., Cell Div 2011; 6:18; and Nguyen, C L et al., Mol CellBiol 2012; 32:3963-77).

siRNAs to several Src family kinases (SFKs) or related signalingproteins (FYN, TXK, CAM2 KB) also reduced viability in one or more HNSCCcell lines and were prioritized as candidates in the cross speciescomparisons. FYN is a SFK that is involved in many pro-oncogenic processsuch as cell proliferation, integrin-mediated and PI3K signaling, whileTXK is a tyrosine kinase that is activated by the SRC family kinase LYN(Xi, S. et al., J Biol Chem 2003; 278:31574-83; Debnath, J. et al., MolCell Biol 1999; 19:1498-507; and Mano, H. et al., FASEB J 1996;10:637-42). SFKs are activated by mitogenic signals to induce HNSCC cellproliferation and LYN mediates cell motility and tumor growth in headand neck cancer (Mano, H. et al., FASEB J 1996; 10:637-42; and Wheeler,S E et al., Clin Cancer Res 2012; 18:2850-60). Furthermore, SRC/FAKsignaling correlates strongly with phenotypes associated with tumorprogression such as invasion and metastasis (Wheeler, S E et al., ClinCancer Res 2012; 18:2850-60; and Zhang, Q., et al. Cancer Res 2004;64:6166-73) and FAK itself is amplified in HNSCC (Agochiya, M. et al.,Oncogene 1999; 18:5646-53), providing further support for targeting theSFK pathway in more aggressive subtypes of HNSCC (Egloff, A M et al.,Semin Oncol 2008; 35:286-97; and Stabile, L P et al., Clin Cancer Res2013; 19:380-92).

In summary, our cross-species functional kinomic approach usingautologous pairs of primary and recurrent/metastatic p53 mutant HNSCClines, coupled with isogenic mouse SCC cells with defined mutationsalong the p53 pathway has identified several survival kinases ascandidate therapeutic targets for aggressive HNSCC. These kinasesregulate a range of cellular processes such as phosphatidylinositol,focal adhesion, and Src signaling pathways, and the G2/M cell cycletransition, suggesting functional targets for therapeutic intervention.Discovery and development of multiple targets may prove to be a usefulstrategy, as tumors frequently develop resistance to single agents andtargeting multiple vulnerabilities simultaneously may be a required toachieve long term remission.

Our preclinical data on WEE1 illustrates not only the vulnerabilities ofp53 mutant HNSCC cells to deregulation of G2/M transition, but alsosupports the initiation of clinical trials with MK-1775 or other G2/Mcheckpoint inhibitors for HNSCC, particularly in combination withcisplatin. More generally, this study illustrates the utility ofintegrating functional genomic approaches with more traditionaldescriptive genomic and molecular profiles to identify therapeutictargets in cancer.

Example 2 Validation of Therapeutic Drug Targets for Tumor SuppressorMutant Cancers in Human Patients

To confirm the kinases identified in Example 1 as therapeutic drugtargets in human cancers, we performed large scale siRNA screens, usingprimary tumor cell cultures obtained from patients with a mutation in atumor suppressor gene. These studies validated kinases identified inExample 1 as therapeutic candidates in primary human tumors. Certainscreens were performed using siRNAs to 6,650 genes considered druggable,i.e., the “druggable genome.” One screen was performed using a customdesigned siRNA library (1302 genes) that included the human kinome (713genes), DNA damage and repair genes (318 genes), and a Kemp PancreaticOncolibrary that includes apoptotic and autophagic genes (96 genes),pancreatic-specific biomarkers (81 genes) (Harsha et al., 2009),pancreatic-specific extracellular/membrane associated genes from serialanalysis of gene expression (44 genes) (Jones et al., 2008), pancreaticcancer pathway controls (44 genes), and chromatin-modifying enzymes (6genes).

We performed a large scale siRNA screen, using 6,659 siRNAs, on aprimary tumor cell culture derived from a patient with head and necksquamous cell carcinoma. This patient had a mutation in p53 (P278S).Among the 38 kinases identified in the HNSCC cell line screens, nine(WEE1, CHEK1, GK2, PIP5K1B, EPHA2, RPS6KL1, MPP3, EPHA3, AURKA) wereidentified as “hits” in the primary culture as well, since inhibition ofthese genes with siRNAs caused growth arrest of primary cultures ofcells obtained from this p53 mutant head and neck squamous cellcarcinoma tumor. This further validates the evidence for these ninegenes being therapeutic targets in human cancers.

We also performed a large scale siRNA screen, using 6,659 siRNAs, on aprimary tumor cell culture derived from a patient with pancreaticcancer. This patient had mutations in both KRAS and p53. The followingkinases identified in the HNSCC cell line screens, WEE1, CHEK1, CAMK2B,and GK2, were also identified as “hits” in this primary culture, sinceinhibition of these genes with siRNAs caused growth arrest of primarycultures of cells from KRAS and p53 mutant pancreatic cancer. Thisfurther validates these genes as therapeutic targets.

We also performed a larger scale siRNA screen, using 1,302 siRNAs, on aset of seven KRAS mutant pancreatic cancer cell lines and one KRAS WTnormal pancreatic ductal epithelial cell line. siRNAs to the followingkinases were preferentially lethal to KRAS mutant pancreatic cancercells as compared to normal cells: FGFR3, NUAK2, ABL1, NEK4, AURKA,WEE1, CHEK1. This further validates these genes as therapeutic targets,since inhibition of these genes with siRNAs caused growth arrest onprimary cultures cells from KRAS mutant pancreatic cancer.

These studies demonstrated that therapeutic targets identified inExample 1 are also targets in primary cell cultures of cells obtainedfrom human cancer patients.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

1. A method for treating or preventing a cancer in a subject in needthereof, wherein the cancer comprises a mutation in a tumor suppressorgene, the method comprising providing to the subject an inhibitor of akinase selected from NEK4, TRIB2, TXK, CAMK2B, NUAK2, EPHA3, ALK,PIP5K1B, RPS6KL1, FGFR3, WEE1, and PRKCE.
 2. The method of claim 1,wherein the tumor suppressor gene is a DNA-PKcs, Atm, p19^(Arf), p53,Hras, Kras or Prkdc gene, or wherein the cancer comprises a mutation inboth a p53 tumor suppressor gene and one or more of a Hras or Kras gene.3.-11. (canceled)
 12. The method of claim 1, wherein the cancer is aprimary tumor.
 13. The method of claim 1, wherein the cancer is arecurrent or metastatic tumor. 14.-15. (canceled)
 16. The method ofclaim 1, wherein the kinase is WEE1, and the subject is further providedwith cisplatin.
 17. The method of claim 1, further comprisingdetermining a level of expression or activity of the kinase or the geneencoding the kinase in cancer cells obtained from the subject.
 18. Themethod of claim 17, wherein the level of expression or activity of thekinase gene or its encoded gene product is determined prior to providingthe subject with the inhibitor.
 19. A method of monitoring theprogression or regression of a tumor in response to a treatment,comprising: (a) determining a level of expression or activity of akinase gene or its encoded gene product in cancer cells obtained fromthe subject prior to or at about the same time as providing the subjectwith the inhibitor; and (b) determining a level of expression oractivity of the kinase gene or its encoded gene product in cancer cellsobtained from the subject following a time period after providing thesubject with the inhibitor; wherein a reduced level of expression oractivity determined for (b) as compared to the level of expression oractivity determined for (a) indicates that the treatment is effective,and wherein the kinase is NEK4, TRIB2, TXK, CAMK2B, NUAK2, EPHA3, ALK,PIP5K1B, RPS6KL1, FGFR3, WEE1, or PRKCE.
 20. A method of identifying atherapeutic drug target in a diseased cell, comprising: (a) screening aplurality of siRNAs for their ability to inhibit growth or reduceviability of a human diseased cell or a human cell having a definedmutation; (b) identifying one or more genes targeted by one or moresiRNAs of (a) that inhibit growth or reduce viability of the human cell;(c) screening a plurality of siRNAs for their ability to inhibit growthor reduce viability of a non-human diseased cell or a non-human cellhaving a defined mutation; (d) identifying one or more genes targeted byone or more siRNAs of (c) that inhibit growth or reduce viability of thenon-human cell; and (e) determining one or more genes identifiedaccording to both (b) and (d), wherein the human diseased cell and thenon-human disease cell share the same disease or the mutation in thehuman cell is in the same gene as the mutation in the non-human cell,and wherein the one or more genes determined according to (e) or theirencoded products are identified as therapeutic drug targets. 21.-32.(canceled)